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Development of an ELISA for the Detection of Azaspiracids Ingunn A. Samdal, Kjersti E. Løvberg, Lyn Ruth Briggs, Jane Kilcoyne, Jianyan Xu, Craig J. Forsyth, and Christopher Owen Miles J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.5b02513 • Publication Date (Web): 06 Aug 2015 Downloaded from http://pubs.acs.org on August 14, 2015

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Journal of Agricultural and Food Chemistry

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Development of an ELISA for the Detection of

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Azaspiracids

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Ingunn A. Samdal†,*, Kjersti E. Løvberg†, Lyn R. Briggs‡, Jane Kilcoyne§, Jianyan Xu#,

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Craig J. Forsythζ, and Christopher O. Miles†

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†

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‡

Norwegian Veterinary Institute, P.O. Box 750 Sentrum, N-0106 Oslo, Norway AgResearch, Ruakura, East Street, Private Bag 3123, Hamilton, New Zealand §

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Marine Institute, Rinville, Oranmore, County Galway, Ireland

#

Department of Chemistry, University of Minnesota-Twin Cities, Minneapolis, MN, 55455, USAa

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ζ

Department of Chemistry and Biochemistry, The Ohio State University, Columbus, OH,

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43220, USA

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a

Present address: Shanghai Hengrui Pharmaceutical Co., LTD, No. 279 Wenjing Road,

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Minhang District, Shanghai City, P.R.China

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*Author to whom correspondence should be addressed:

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Phone +47 2321 6226

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Fax

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Email [email protected]

+47 2321 6201

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ABSTRACT

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Azaspiracids (AZAs) are a group of biotoxins that cause food poisoning in humans. These

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toxins are produced by small marine dinoflagellates such as Azadinium spinosum, and

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accumulate in shellfish. Ovine polyclonal antibodies were produced and used to develop an

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ELISA for quantitating AZAs in shellfish, algal cells, and culture supernatants. Immunizing

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and plate coating antigens were prepared from synthetic fragments of the constant region of

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AZAs. The ELISA provides a sensitive and rapid analytical method for screening large

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numbers of samples. It has a working range of 0.45−8.6 ng/mL and limit of quantitation for

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total AZAs in whole shellfish at 57 µg/kg, well below the maximum permitted level set by the

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European Commission. The ELISA has good cross-reactivity to AZA-1 to 10, 33, 34 and 37-

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epi-AZA-1. Naturally contaminated Irish mussels gave similar results whether they were

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cooked or uncooked, indicating that the ELISA also detects 22-carboxyAZA metabolites (e.g.

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AZA-17 and AZA-19). ELISA results showed excellent correlation with LC-MS/MS analysis,

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both for mussel extract spiked with AZA-1 and for naturally contaminated Irish mussels. The

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assay is therefore well suited to screening for AZAs in shellfish samples intended for human

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consumption, as well as for studies on AZA metabolism.

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KEYWORDS: Azaspiracid; AZA-1; ELISA; Immunoassay; Antibody; Polyclonal; Shellfish

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toxin; Mussel

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INTRODUCTION

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The first poisoning incident associated with azaspiracids (AZAs) occurred in November 1995,

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when at least eight people in the Netherlands became ill after eating mussels (Mytilus edulis)

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harvested at Killary Harbor, Ireland.1 Symptoms included nausea, vomiting, severe diarrhea,

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and stomach cramps. Although these symptoms are similar to those of diarrhetic shellfish 2 ACS Paragon Plus Environment

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poisoning (DSP), levels of the major DSP toxins okadaic acid and dinophysistoxins were very

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low.1 Satake et al.2 isolated and identified the first AZA analog and named it azaspiracid, now

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referred to as AZA-1 (Figure 1). In November 1997, the toxicity recurred in mussels from

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Aranmore Island, Ireland, and new human intoxications were reported.3 New analogs of AZA

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were identified (AZA-2–5).4 AZAs are natural products differing from previously known

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nitrogen-containing toxins found in shellfish or dinoflagellates by having two unique spiro

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ring assemblies, a cyclic amine and a carboxylic acid.2, 4 Since its initial report, the structure

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of AZAs has been revised.5, 6 The revised structures are shown in Figure 1 and ~30 AZAs

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have been reported.7

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Since their initial discovery, AZAs have been found in shellfish harvested throughout Europe,

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including mussels in Spain,8 England and Norway,9, 10 in mussels, oysters, clams and cockles

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in Ireland,11 in brown crabs from Sweden and Norway12 and in scallops from France.8, 13

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AZAs have also been reported in shellfish from north-west Africa,14 Canada,15 Chile,16, 17 and

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China,18 and in a sponge from Japan.19 The search for a causative alga led to the identification

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of the dinoflagellate Azadinium spinosum that was reported to be the source of AZA-1 and

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AZA-2,20, 21 and several other species of dinoflagellates have subsequently been shown to

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produce AZAs.22, 23

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The EU has set a maximum level of AZAs in whole uncooked shellfish destined for human

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consumption at 160 µg/kg AZA-equivalents24 and specifies AZA-1, AZA-2 and AZA-3.25

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The reference method for the analysis of AZAs is LC-MS/MS.25 Chemical methods require

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sophisticated instrumentation and highly trained personnel, and are therefore relatively

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expensive to perform. Detection of AZAs by enzyme-linked immunosorbent assay (ELISA) is

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well suited to rapid screening because immunoassays can be highly sensitive and it is not

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necessary to possess all AZA analogs as standards, provided the assay cross-reactivity is

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similar towards all analogs. Furthermore, immunoassays can often be reformatted to produce 3 ACS Paragon Plus Environment


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biosensors and dipstick assays. So far, only two antibodies to AZAs have been reported: a

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polyclonal antibody raised using a synthetic fragment of AZA,26 and monoclonal antibodies

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raised using AZA-1.27 Recently, one of these monoclonal antibodies was used in a

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microsphere-based immunoassay.28

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Here we report the use of the polyclonal antibody against AZAs,26 after further immunisations

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of the same sheep, to develop the first ELISA method for quantitation of these compounds in

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mussels at concentrations below the current regulatory limit.

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MATERIALS AND METHODS

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Materials. AZA-1 was from the Marine Institute, Ireland.29 Bovine serum albumin (BSA),

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ovalbumin (OVA), L-cysteine, bromine (Br2) and Freund’s incomplete adjuvants were from

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Sigma Aldrich (St. Louis, MO). Cationized BSA (cBSA) was prepared by standard

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methods30. Maxisorp immunoplates (96 flat-bottom wells) were from Nunc (Roskilde,

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Denmark), poly(vinylpyrrolidone) 25 (PVP) was from Serva Electrophoresis (Heidelberg,

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Germany), rabbit–anti-sheep horseradish peroxidase conjugate (anti–sheep-HRP) from

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Zymed (now Invitrogen, Paisley, UK), and the HRP-substrate K-blue Aq. from Neogen

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(Lexington, KY). Certified reference materials (CRMs) of AZA-1, AZA-2, AZA-3,

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microcystin-LR, and okadaic acid were from the National Research Council of Canada

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(Halifax, NS, Canada). Quantitative laboratory reference materials (LRMs) of AZA-4–10,

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AZA-33, AZA-34 and 37-epi-AZA-1 were prepared as described by Kilcoyne et al.31-33

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Pinnatoxin G was from Cawthron Institute (Nelson, New Zealand). All other inorganic

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chemicals and organic solvents were of reagent grade or better. Plate-coating buffer was

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carbonate buffer (50 mM, pH 9.6). Phosphate-buffered saline (PBS) contained NaCl (137

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mM), KCl (2.7 mM), Na2HPO4 (8 mM), and KH2PO4 (1.5 mM), pH 7.4. ELISA washing 4 ACS Paragon Plus Environment

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buffer (PBST) was 0.05% Tween 20 in PBS. Sample buffer was 10% MeOH (v/v) in PBST

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and the antibody buffer consisted of 1% PVP (w/v) in PBST.

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Immunogen. The synthetic AZA-hapten-1 was prepared and conjugated to cBSA as

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described by Forsyth et al.26 Hapten-2 was prepared by a novel synthetic route34 and

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conjugated to cBSA in the same manner as for hapten-1 to form cBSA-hapten-2 (Figure 2).

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The immunogen was washed through multiple centrifugations with PBS in a Vivaspin 5.0 mL

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concentrator with 10 000 MW cutoff (Sartorius Stedim Biotech GmbH, Goettingen,

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Germany) to remove unreacted hapten and excess reagents. The cBSA-hapten-2 was

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dispensed with aliquots (1 mg in 100 µL), lyophilized, and stored at −20 °C.

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Plate Coating Antigen (OVA–BrAZA-1). Purified AZA-1 (40 µg) was dissolved in MeOH

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(100 µL), to which was added 50 µL of bromine solution (5 µL Br2 in 10 mL MeOH). After 1

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h the solution was evaporated to dryness under a stream of nitrogen to remove solvent and

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residual bromine. The residue was dissolved in MeOH (100 µL) and added to a solution of

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ovalbumin (5 mg) in coating buffer (1 mL). After reaction for 18 h, 1 mL of 5 mg/mL L-

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cysteine in PBS was added to quench unreacted bromides for 2 h at 4 °C. The OVA-BrAZA-1

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was washed, dispensed as aliquots (1 mg in 405 µL), lyophilized, and stored as for the

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immunogen.

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Polyclonal Antibodies. Six sheep were immunized, three in New Zealand (Romney breed)

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and three in Norway (Nor X breed). The first six immunizations were with cBSA–hapten-1

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(Figure 2),26 followed by five immunizations with cBSA–hapten-2. During these 11

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immunizations, the sheep were given three long resting periods to mature the immune

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response. Immunizations in New Zealand were, in general, performed as described by Briggs



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et al.,35 (300 µg of immunogen in 1.0 mL water-in-oil emulsion) and in Norway as described

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by Samdal et al.36

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ELISA. Maxisorp immunoplates were coated overnight at ambient temperature with plate

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coating antigen OVA–BrAZA-1 in coating buffer (100 µL/well) at 10 µg/mL. After

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incubation, the plates were washed four times with PBST. The plates were blocked for 1 h

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with 1% PVP in PBS (300 µL/well), then washed twice with PBST.

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Non-competitive assays were used to estimate titers of the sera giving a maximum absorbance

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of 1.0. This was done by combining equal volumes (50 µL) of sample buffer (10% MeOH in

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PBST) and dilution series of antisera in antibody buffer (1% PVP in PBST) in wells and

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incubating for 1 h. After washing four times with PBST, bound antibody was detected by

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adding anti-sheep–HRP conjugate diluted 1:3000 in antibody buffer (100 µL/well) for 2 h,

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then washing four times before addition of the HRP substrate (100 µL/well). After 15 min, the

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reaction was stopped by adding 50 µL 10% H2SO4 and absorbances measured at 450 nm

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using a model 1420 Victor2 plate reader (Wallac, Turku, Finland). All incubations were

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performed at ~ 20 °C.

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Competitive ELISAs were performed as described above, by adding appropriate amounts of

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standard or sample, and antiserum added to the wells after blocking. Concentrated standards

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in MeOH, usually AZA-1 (1.31 µg/mL), were diluted in PBST to give a MeOH concentration

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of 10%, and then in a three-fold dilution series in sample buffer, giving standard

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concentrations of 0.0066 to 131 ng/mL. Shellfish extracts in MeOH were similarly diluted 10

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times with PBST to adjust for the MeOH concentration, followed by a dilution series in

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sample buffer. All sample and standard dilutions were analyzed in duplicate wells. The

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remaining ELISA steps were as described for the non-competitive ELISA. 6 ACS Paragon Plus Environment

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Optimization. Optimal concentrations of plate-coating antigen (10 µg/mL), antiserum

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AgR367-11b (1:8700), and anti-sheep–HRP (1:3000) were determined by checkerboard

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titrations followed by optimization of the standard curve. Assay standard curves were

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calculated using logistic treatment of the data. The assay working range was defined as the

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linear region at 20−80% of maximal absorbance (Amax).

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Cross-Reactivity. The relative specificity of the immunoassay toward the available AZAs,

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microcystin-LR, okadaic acid and pinnatoxin G was determined by assaying a dilution series

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of each compound in sample buffer. The I50 values (molar concentration giving 50%

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inhibition) are expressed as a percent I50 relative to the AZA-1 CRM.

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LC-MS/MS Analysis. Quantitation of AZA1–3 and -6 was performed on a 2695 LC coupled

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to a Micromass triple-stage quadrupole (TSQ) Ultima (both from Waters, Manchester, UK)

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operated in multiple reaction monitoring (MRM) mode, with the following transitions: AZA1

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m/z 842.5→654.4 and 842.5→672.4, AZA2 856.5→654.4 and 856.5→672.4, AZA3

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828.5→640.4 and 828.5→658.4, AZA6 842.5→640.5 and 842.5→658.4. The cone voltage

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was 60 V and the collision voltage was 40 V, the cone and desolvation gas flows were set at

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100 and 800 L/h, respectively, and the source temperature was 150 °C.

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Binary gradient elution was used, with phase A consisting of water and phase B of 95%

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acetonitrile in water (both containing 2 mM ammonium formate and 50 mM formic acid).

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Chromatography was performed with a 50 mm × 2.1 mm i.d., 3 µm, Hypersil BDS C8

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column, with a 10 mm × 2.1 mm i.d. guard column of the same material (Thermo Scientific,

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Waltham, MA). The gradient was from 30% B, to 90% B over 8 min at 0.25 mL/min, held for

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5 min, then held at 100% B at 0.4 mL/min for 5 min, and returned to the initial conditions and 7 ACS Paragon Plus Environment


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held for 4 min to equilibrate the system. The injection volume was 5 µL and the column and

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sample temperatures were 25 °C and 6 °C, respectively.

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Mussel Extracts. AZA-contaminated raw mussel (M. edulis) samples, tested as part of the

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routine monitoring programme in Ireland, were selected for analysis. The shellfish were

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shucked and homogenised using a Waring blender before extraction. Tissue samples were

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weighed (2 g) and duplicate samples sealed in 50 mL centrifuge tubes. One sample was

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placed in a water bath and heated to 90 °C for 10 min and then allowed to cool. Both samples

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were extracted by vortex mixing for 1 min with 9 mL of MeOH, centrifuged at 3,950 g (5

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min), and the supernatants decanted into 25 mL volumetric flasks. The remaining pellet was

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further homogenized and extracted using an Ultra Turrax (IKA, Staufen, Germany) for 1 min

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with an additional 9 mL of MeOH, centrifuged at 3,950 g for 5 min, and the supernatant

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decanted into the same 25 mL volumetric flask, which was then brought to volume with

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MeOH. Extracts were analyzed by LC-MS/MS and ELISA.

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Spiked Tissue Extracts. A mussel extract that had trace levels of AZAs (< 0.1 ng/mL by LC-

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MS/MS) was spiked with an AZA-1 CRM at 6 concentrations ranging from 5 to 200 ng/mL.

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RESULTS AND DISCUSSION

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Hapten synthesis, conjugation and immunogen preparation. A critical step in designing

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hapten chemistry to generate an immunoassay with appropriate specificity is deciding which

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sub-structures within the analyte should be recognized by the antibodies, and which should

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not be recognized. Just as important, when dealing with numerous variants of AZA with

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modifications at several sites on the parent molecule, is to balance the preference of the

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antibodies for particular toxin variants in order to achieve uniform cross-reactivity. 8 ACS Paragon Plus Environment

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Most AZAs have very similar structures, with all of the published analogs differing in the

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C3−C16 area and at C-22, C-23 and C-37 in the molecule (Figure 1). Therefore, with the aim

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of developing an ELISA with broad specificity to AZAs, the structurally conserved region C-

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26–C-40 appeared to be a favorable area to raise antibodies against. Furthermore, the scarcity

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of AZA-1 at the time, and the development of several partial syntheses of AZAs, made the

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constant C26–C40 area of the AZAs even more attractive as an immunogen. Subsequent

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attempts at raising antibodies against AZA-1 itself have encountered problems with toxicity.27

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Two similar haptens were synthesized (Figure 2), representing the major structural elements

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of the AZA C26–C40 region (Figure 1). Hapten-1, an AZA fragment with a ketone at the

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position corresponding to C-26 in AZA-1, was synthesized and initially used for

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immunization.26 Later, hapten-2, with an olefinic methylene instead of the ketone in hapten-1,

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was synthesized to resemble AZAs more closely,34 and used to continue the immunization

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program. Both haptens were conjugated to cBSA in order to make them immunogenic. Both

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immunogens present the structurally conserved region of AZAs (C-26–C-40) to the sheep’s

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immune system, and it was hoped that this would result in antibodies specific to the C-26–C-

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40 moiety and therefore with good recognition for the wide range of known AZA variants.

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Plate coating antigen. For small molecules such as AZAs, ELISA format options are limited

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to competitive assays, where the antibody can bind either to the AZA on the plate-coating

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antigen (or on a reporter molecule) or to free AZAs in the sample. Because the assay is based

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on competition between the plate-coating antigen and the analyte for the antibody’s binding

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sites, the plate coating chemistry is important when developing a competitive immunoassay,

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especially with polyclonal antisera. During assay development, several plate-coating antigens



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were prepared and tested. These initially used hapten-1, later hapten-2, and finally BrAZA-1,

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bound to OVA.

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Precharacterization of AZA-antibodies. Between immunizations, antisera were screened by

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indirect ELISA for AZA-specific antibodies initially using OVA–hapten-1 and later OVA–

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hapten-2. Hapten-specific antibodies were detectable in each antiserum after the first booster

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injection. Antisera titers were determined by non-competitive ELISA. Antiserum from sheep

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AgR367 gave the highest antibody titer (1:200,000) after four immunizations, whereas

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antisera from other sheep had much lower (5–200-fold) titers. Initial screening for

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competition, by addition of AZA-1, showed that acceptable standard curves could only be

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obtained using antiserum AgR367 (I50 at 160 ng/mL). Therefore this antiserum was used for

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assay development and optimization.

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ELISA optimization. The ELISA was optimized with 10% MeOH in all samples and

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standards, in order to be compatible with standard methods for extraction and analysis of

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mussels for lipophilic algal toxins. To obtain maximum ELISA sensitivity, the assay

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conditions, such as the concentration of reagents, the blocking agent, and temperature need to

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be optimized. Optimal concentrations of assay reagents were determined by checkerboard

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titrations and standard curves. Criteria used to evaluate optimization were Amax, working

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range (I20−I80), I50, slope of the curve, and limit of quantitation (LOQ; I20 based on the mean

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of several assays and multiplied by the dilution factor (i.e., 10 for MeOH-extracts)). Note that

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AZAs in mussel tissue were diluted 12.5-fold during extraction with 100% MeOH, and the

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LOQ for AZAs in mussel tissue is therefore the assay I20 multiplied by 12.5 and then 10.

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Assay optimization was carried out using bulk collections of antisera taken from sheep

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AgR367 after four, six, ten and eleven immunizations (AgR367-4b, -6b, -10b and -11b). 10 ACS Paragon Plus Environment

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Assay sensitivity was increased by using 1% PVP as the blocker, rather than 1% BSA, 1%

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OVA, 1% Superblock or 1% Casein. Furthermore, use of PVP as the blocker resulted in up to

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5-fold reduction in the amount of antiserum required for the assay.

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Assay sensitivity and competition were improved by changing the plate-coating antigen from

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OVA–hapten-1 to OVA–hapten-2. The working range improved from 17−1054 to 4−62

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ng/mL, and the sensitivity (I50) from 135 to 16 ng/mL, increasing assay sensitivity

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approximately 8-fold (using AgR367-4b). With OVA–BrAZA-1 as plate coating antigen, the

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optimized assay (average of 13 assays) had a working range (I20−I80) of 0.45−8.6 ng/mL and

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the I50 was 1.9 ng/mL. This change to OVA–BrAZA-1 (and AgR367-11b) made the assay 8-

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fold more sensitive than with OVA–hapten-2 and 71-fold more sensitive than with OVA–

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hapten-1. This improved sensitivity may be due the antibodies’ affinity for OVA–BrAZA-1

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being closer to that for free AZAs (the analyte) than was the case for OVA-hapten-1 and

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OVA–hapten-2. This would be expected to lead to more effective competition by the analytes,

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and thus higher assay sensitivity for AZAs. Standard curves obtained with the CRMs of AZA-

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1, AZA-2 and AZA-3 are shown in Figure 3.

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Specificity. Reduction in the competitive ELISA signal upon addition of AZA-1 indicates

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competition, but does not show which other target analyte can bind to the antibodies. Often,

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ELISAs are aimed at recognizing one compound specifically. In this case, we aimed to

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develop an assay recognizing all AZAs containing the right-hand part of the AZA structure

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(C-26−C-40). Therefore, the response of the optimized assay to AZA-1−10, AZA-33, AZA-

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34 and 37-epi-AZA-1 (Figure 1), all of which are reported to be toxic33, was tested over a

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range of concentrations to determine cross-reactivity (Table 1). All AZA standards caused

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concentration-dependent inhibition of antibody binding. Based on I50 values, the molar cross11 ACS Paragon Plus Environment


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reactivities relative to AZA-1 were 75−140% for the CRMs of AZA-1, AZA-2 and AZA-3,

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and 57−145% for LRMs of the remaining AZAs (Table 1). The antibodies in the assay

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therefore have a similar affinity for these other AZAs as they do for the reference standard,

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AZA-1. Similar tests with microcystin-LR (50 ng/mL), okadaic acid (1.4 µg/mL) and

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pinnatoxin G (1.0 µg/mL) resulted in no inhibition. Cross-reactivity to these toxins is less

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than 1 % with the antibody, indicating that the antibody binds specifically to AZAs.

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Cross-reactivity studies indicated that recognition of analogs by antibodies in the ELISA was

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slightly reduced for AZA-2, AZA-7, AZA-33 and 37-epi-AZA-1 (Table 1), whereas it was

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slightly increased for AZA-3, AZA-4, AZA-6 and AZA-10. This may indicate that

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substitution of the methyl group at C-22 (R3) for hydrogen slightly favors binding to the

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antibody, although AZA-5 does not show the same increase. The substituents at C-3, C-8 and

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C-23 (R1, R2 and R4) seem to have less influence on antibody binding, which is as expected

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given that these substituents are not part of the C-26–C-40 present in the immunogens. A lack

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of standards of the other AZAs prevented characterization of the cross-reactivity of AZA-11

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to AZA-32, but it is anticipated that the antibodies will recognize most of the known analogs,

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since they all contain the C-26−C-40 region of the AZA (Figure 1). Furthermore, that the

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ELISA gave essentially identical results from cooked and uncooked mussels suggests that the

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22-carboxy congeners AZA-17, -19, -21, and -23 also have similar cross-reactivities to their

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corresponding thermal degradation products (AZA-3, -6, -4 and -9, respectively)37. Cross-

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reactivity with AZA-33 (57%) confirms that rings A–D are not included in the antibody

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binding site.

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37-epi-AZA-1 is identical to AZA-1 except for a stereochemical change at C-37, where the

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methyl group is orientated axially (37R) instead of equatorially (37S) as in all the other AZA

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analogs. The ELISA cross-reactivity was 77% for 37-epi-AZA-1. This structural change is in 12 ACS Paragon Plus Environment

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the “constant region” and may explain the slightly lower response, but nevertheless suggests

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that antibody binding is not especially sensitive to substitution at C-37.

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The ELISA therefore appears to detect all of the reported AZA congeners. It should be noted,

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however, that although the assay estimates the total content of AZAs, this does not necessarily

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reflect the toxicity of the sample because different congener structures vary in toxicity.

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Standards. The availability of reliable standards is critical for the development and validation

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of analytical methods. One obstacle in this study was the relatively limited array of AZA

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analogs that were available for cross-reactivity studies. The criteria for what constitutes a

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good standard may vary between analytical methods. For example, standards adequate for

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LC-MS may contain contaminating analogs which do not interfere with quantitation due to

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differences in mass and retention time. However, for antibody-based detection, contaminating

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AZA analogs can affect the response. Even for highly pure standards, all analytical methods

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require that standards are quantitatively accurate in order to produce reliable results.

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Consequently, cross-reactivity data should be regarded only as indicative, except where high-

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purity quantitatively verified standards have been used. When calculating cross-reactivities

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for the various AZA standards, the known impurities in the CRMs and LRMs were corrected

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for. However, because the AZA standards were relatively pure, these corrections resulted in

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only minor changes to the calculated cross-reactivities in Table 1.

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Preliminary validation. Preliminary investigations with methanolic extracts from mussels,

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scallops, oysters and algal extracts showed minimal matrix effects (data not shown). The

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LOQ for total AZAs in whole shellfish was calculated to be 57 µg/kg (2 g extracted with 25

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mL MeOH).

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AZA-1 was spiked into extracts of a natural sample of Irish mussel previously shown by LC-

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MS to contain only trace levels of AZAs. The sample was spiked with six concentrations of

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AZA-1 (Figure 4A). Analyses determined by the optimized ELISA and LC-MS/MS were

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compared with the calculated AZA-1 concentrations. The results for AZAs by ELISA and for

333

AZA-1 by LC-MS/MS were very similar (Figure 4A) and the ELISA correlates well with LC-

334

MS/MS (within ± 20%) between 10 and 150 ng/mL (corresponding to 125–1875 µg/kg in

335

whole shellfish). Both methods, however, appeared to underestimate the levels for very high

336

concentrations (12–16 times the regulatory level of AZA-1). The average recovery by ELISA

337

was 102%, well within the acceptable limits for assay recovery (100% ± 20).

338 339

To confirm its applicability to real samples, the ELISA was used to analyze AZAs in 13

340

shellfish samples (Mytilus edulis). Of these 13 samples, all were extracted as raw tissues and

341

analyzed, whereas 11 were also extracted as cooked shellfish tissues, without changing the

342

water content, and analyzed (Figure 4B and C). For comparison, all samples were also

343

analyzed by ELISA for AZAs and by LC-MS for AZA-1, AZA-2, AZA-3 and AZA-6. Figure

344

4B shows results for all samples (13 raw and 11 cooked), whereas Figure 4C shows only

345

results up to around the regulatory level of 160 µg/kg24 that corresponds to 12.8 ng/mL in the

346

extracts (8 raw and 6 cooked samples). ELISA results were ~ 2-fold those obtained by

347

analysis with LC-MS/MS for AZA1–3 and AZA-6. Presumably this is due to the presence of

348

minor AZAs that were not targeted in the LC-MS/MS analysis, since LC-MS/MS and the

349

ELISA showed an excellent 1:1 correlation for AZA-1-spiked shellfish. Results by ELISA

350

were essentially identical regardless of whether the shellfish had been cooked or not, whereas

351

LC-MS/MS results were ~ 20% lower for uncooked shellfish. This difference is attributable to

352

the heat-promoted conversion of the mussel metabolites AZA-17 and AZA-19 (not analysed

353

for by LC-MS/MS), present in the uncooked tissue, into AZA-3 and AZA-6 (which were


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354

analyzed for) by cooking, and suggests that the antibodies recognize AZA-17 and AZA-19

355

with approximately the same cross-reactivity as for AZA-3 and AZA-6.

356 357

The extraction method used for extracting azaspiracids from mussels was unfortunately not

358

well suited to the AZA-ELISA. Extraction with a lower volume of solvent, and containing a

359

higher percentage of water (so that a smaller dilution of the extract was required), would be

360

expected to considerably improve the LOQ of the ELISA for total AZAs in mussels.

361 362

In summary, we report development of the first ELISA for the detection of AZAs. The

363

antibodies were developed using two synthetic fragments of AZA, respectively hapten 1, as

364

reported previously26, and with further immunizations with conjugates of the improved

365

hapten-2.34 In this report we have applied the resulting antibodies to development of an AZA-

366

ELISA. This ELISA is specific for AZAs, with similar cross-reactivity to a broad range of

367

AZA analogues, so that the total content of analogs and metabolites can be measured. The

368

antiserum together with the plate-coating antigen (OVA–BrAZA-1) provides an ELISA with

369

good sensitivity and broad specificity.

370 371

This ELISA for AZAs is a sensitive and rapid analytical method that uses inexpensive

372

instrumentation and is suitable for use as a tool for shellfish toxin analysis. The broad

373

specificity of the antibodies means that the ELISA provides an estimate of the total content of

374

AZA analogs present in the sample. The AZA-ELISA detects all the analogs that the

375

European Commission25 currently require testing for, i.e., AZA-1, AZA-2 and AZA-3 (with

376

cross-reactivities of 100, 75 and 140 %, respectively). In addition, the ELISA can also detect

377

a wide range of other AZAs, including AZA-17 and AZA-19, which are converted to AZA-3

378

and AZA-6 respectively during cooking. Thus, the assay detects all the regulated AZAs, as

379

well as some of their precursors, and in addition it detects other AZA metabolites such as 15 ACS Paragon Plus Environment


380

AZA-4, AZA-5, and AZA-7–10 which are also known to be toxic.32 Comparison of the

381

ELISA and LC-MS should facilitate detection of new metabolites of AZAs in a similar

382

manner as reported for yessotoxins.38

383 384

ASSOCIATED CONTENT

385

Supporting Information

386

ELISA inhibition curves on three different coaters, molar cross-reactivities of antiserum

387

AgR367-11b with a series of AZA analogs (extended version), and tabulated impurities in the

388

AZA-standards. This material is available free of charge via the Internet at http://pubs.acs.org.

389 390 391

AUTHOR INFORMATION

392

Corresponding Author

393

* Tel.: +47 2321 6226. Fax: +47 2321 6201. E-mail: [email protected]

394 395

Funding

396

This study was supported by grant 139593/140 from the Norwegian Research Council, by the

397

BIOTOX project (partly funded by the European Commission, through the 6th Framework

398

Programme contract no. 514074, priority Food Quality and Safety), by the European Union

399

Seventh Framework Programme (FP7/2007–2013) under the ECsafeSEAFOOD project (grant

400

agreement n° 311820), and by Norwegian Veterinary Institute. This study was also supported

401

by the New Zealand Foundation for Research, Science and Technology (International

402

Investment Opportunities Fund, Grant C10X0406. This publication was also made possible

403

by Grant ES10615 from the National Institute of Environmental Health Sciences (NIEHS),

404

NIH, USA (CJF). Its contents are solely the responsibility of the authors and do not

405

necessarily represent the official views of the NIEHS, NIH. Support was also received from 16 ACS Paragon Plus Environment

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406

the Marine Institute and the Marine Research Sub-Programme of the National Development

407

Plan 2007–2013 (PBA/AF/08/001(01)), co-financed under the European Regional

408

Development Fund.

409 410

Notes

411

The authors declare no competing financial interest.

412 413

ACKNOWLEDGEMENTS

414

Sheep for antibody development were provided and cared for by farm staff at AgResearch,

415

Hamilton, New Zealand and The Animal Production Experimental Centre at The Norwegian

416

University of Life Sciences, Ås, Norway. Immunisations and blood sampling was done by

417

assistance from Arne Flåøyen and Jens Børsum at the Norwegian Veterinary Institute and

418

Colleen Podmore (including initial antiserum screening) at AgResearch. All animal

419

experiments were in accordance with the three R’s, laws and regulations, and approved by the

420

AgResearch Ruakura Animal Ethics Committee (Application 11832) or the Norwegian

421

Animal Research Authority. We thank Dr. Y. Ding for synthetic assistance. Standards of

422

AZA-1–3 were kindly provided by M. A. Quilliam at National Research Council of Canada.

423 424

ABBREVIATIONS

425

AZA, azaspiracid; BSA, bovine serum albumin; BrAZA-1, brominated AZA-1; cBSA,

426

cationized BSA; CRM, certified reference material; ELISA, enzyme-linked immunosorbent

427

assay; HRP, horseradish peroxidase; LOQ, limit of quantitation; LRM, laboratory reference

428

material; OVA, ovalbumin; PBS, phosphate-buffered saline; PBST, PBS with 0.05% Tween

429

20; PVP, poly(vinylpyrrolidone) 25.

430



431

REFERENCES

432

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22. Krock, B.; Tillmann, U.; Voß, D.; Koch, B. P.; Salas, R.; Witt, M.; Potvin, É.; Jeong, H.

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26. Forsyth, C. J.; Xu, J. Y.; Nguyen, S. T.; Samdal, I. A.; Briggs, L. R.; Rundberget, T.;

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27. Frederick, M. O.; Marin, S. D.; Janda, K. D.; Nicolaou, K. C.; Dickerson, T. J.,

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34. Xu, J. Progress towards the total synthesis of azaspiracids. PhD thesis, Department of Chemistry, University of Minnesota, Minneapolis, MN, 55455, USA, pp. 34−64, 2006. 35. Briggs, L. R.; Miles, C. O.; Fitzgerald, J. M.; Ross, K. M.; Garthwaite, I.; Towers, N. R.,

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Figure 1. Structures of selected azaspiracids: AZA-1−23 with variable functionality at R1−R4 (C-1, C-8, C-22 and C-23), AZA-33 and AZA-34. Note that 37-epi-AZA-1 differs stereochemically from AZA-1 at position 37.

Figure 2. Structures of hapten-1 and hapten-2 used for preparing immunogens and the initial plate-coating antigen. Both haptens consist of the constant region of AZAs (C-26–C-40).

Figure 3. Standard curves obtained with CRMs of AZA-1, AZA-2 and AZA-3 in the AZAELISA using antibody AgR367-11b on plate coater hapten-2.

Figure 4. Comparison of total AZAs determined by ELISA and LC-MS/MS for samples of mussels (M. edulis). (A) Spiking of a mussel sample with 5, 10, 20, 50, 150 and 200 ng/mL of AZA-1. (B) Analysis of 13 raw (•) and 11 cooked () naturally contaminated samples for AZA-1, -2, -3 and -6 by LC-MS and for total AZAs by ELISA. (C) Expansion of graph B showing the data from 0–16 ng/mL by LC-MS/MS, i.e. close to and under the regulatory limit. The vertical dotted line at 12.8 ng/mL corresponds to the regulatory limit for AZA-1–3 in European shellfish.



Table 1. Molar Cross-Reactivities of a Series of AZA Analogs in the AZA-ELISA Assay range (pg/mL)a % Compound Origin n I20 I80 I50 CRb AZA-1c NRC 13 428 8047 1828 100 AZA-2c NRC 6 504 14462 2666 75 c AZA-3 NRC 6 335 5228 1312 140 AZA-4d MI 3 369 3658 1157 145 AZA-5d MI 4 472 7558 1845 100 d AZA-6 MI 3 315 4595 1195 144 AZA-7d MI 4 446 7642 1824 72 AZA-8d MI 3 431 11202 2173 95 d AZA-9 MI 3 392 6186 1533 114 AZA-10d MI 4 425 6497 1652 128 AZA-33d MI 3 609 13289 2764 57 d AZA-34 MI 3 302 9364 1630 110 37-epi-AZA-1d MI 3 440 15315 2586 77 a I20, I50, I80 are the concentrations of analog giving 20, 50 and 80% inhibition, respectively, of binding of antibody to the coating antigen (OVA–BrAZA-1). All values are corrected for AZA-impurities in the standards. b Molar cross-reactivity; CR = 100 × (I50 AZA-1 CRM)/(I50 analog). Intra-assay variation (CV) was 6−24% for I50-values based on 3−13 competition curves. c CRM. d LRM


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Figure 1. Structures of selected azaspiracids: AZA-1−23 with variable functionality at R1-R4 (C-1, C-8, C-22 and C-23), AZA-33 and AZA-34. Note that 37-epi-AZA-1 differs stereochemically from AZA-1 at position 37. 234x473mm (300 x 300 DPI)

ACS Paragon Plus Environment


Figure 2. Structures of hapten-1 and hapten-2 used for preparing immunogens and the initial plate-coating antigen. Both haptens consist of the constant region of AZAs (C-26–C-40). 78x104mm (300 x 300 DPI)


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Standard curves obtained with CRMs of AZA-1, AZA-2 and AZA-3 in the AZA-ELISA using antibody AgR36711b on plate coater OVA-BrAZA-1.



Comparison of total AZAs determined by ELISA and LC-MS/MS for samples of mussels (M. edulis). (A) Spiking of a mussel sample with 5, 10, 20, 50, 150 and 200 ng/mL of AZA-1. (B) Analysis of 13 raw (•) and 11 cooked (○) naturally contaminated samples for AZA-1, -2, -3 and -6 by LC-MS and for total AZAs by ELISA. (C) Expansion of graph B showing the data from 0–16 ng/mL by LC-MS/MS, i.e. close to and under the regulatory limit. The vertical dotted line at 12.8 ng/mL corresponds to the regulatory limit for AZA-1–3 in European shellfish. 161x381mm (300 x 300 DPI)


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Table of content graphic 259x130mm (72 x 72 DPI)


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