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Development and validation of a lateral flow immunoassay (LFIA) for the rapid screening of okadaic acid and all dinophysis toxins from shellfish extracts Waqass Jawaid, Julie Patricia Meneely, Katrina Campbell, Karrie Melville, Stephen J Holmes, Jennifer Rice, and Christopher T. Elliott J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.5b01254 • Publication Date (Web): 19 Aug 2015 Downloaded from http://pubs.acs.org on August 24, 2015
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Journal of Agricultural and Food Chemistry is published by the American Chemical Society. 1155 Sixteenth Street N.W., Washington, DC 20036 Published by American Chemical Society. Copyright © American Chemical Society. However, no copyright claim is made to original U.S. Government works, or works produced by employees of any Commonwealth realm Crown government in the course of their duties.
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Journal of Agricultural and Food Chemistry
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Development and validation of a lateral flow immunoassay (LFIA) for the rapid screening
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of okadaic acid and all dinophysis toxins from shellfish extracts
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Waqass Jawaida,b,*, Julie P. Meneelyb, Katrina Campbellb, Karrie Melvillea, Stephen J. Holmesa,
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Jennifer Ricec, Christopher T. Elliottb
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a
Neogen Europe Limited, The Dairy School, Auchincruive, Ayr, KA6 5HU, Scotland, UK
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b
Institute for Global Food Security, School of Biological Sciences, Queen’s University, David
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Keir Building, Stranmillis Road, Belfast, BT9 5AG
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c
Neogen Corporation, 620 Lesher Place, Lansing, MI 48912, USA
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* Corresponding author. Tel.: +44 1292 525553; fax.: +44 1292 525601
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E-mail address:
[email protected] (W. Jawaid).
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Abstract
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A single-step lateral flow immunoassay (LFIA) was developed and validated to detect okadaic
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acid (OA) and dinophysis toxins (DTX), which cause diarrhetic shellfish poisoning (DSP). The
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performance characteristics of the test were investigated, in comparison to reference methods
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(LC-MS/MS and/or bioassay), using both spiked and naturally contaminated shellfish. A
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portable reader was used to generate a qualitative result, indicating the absence/presence of OA-
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group toxins, at concentrations relevant to the maximum permitted level (MPL). Sample
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homogenates could be screened in 20 min (including extraction and assay time) for the presence
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of free toxins (OA, DTX1, DTX2). DTX3 detection could be included with the addition of a
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hydrolysis procedure. No matrix effects were observed from the species evaluated (mussels,
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scallops, oysters, clams). Results from naturally contaminated samples (n=72) indicated no false
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compliant results and no false non-compliant results at 90%
190*
130*
68.4%
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Oysters
OA
>90%
280*
350*
125.0%
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Oysters
OA
>90%
410*
230*
56.1%
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Oysters
OA
>90%
440*
700*
159.1%
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Mussels
DTX1
5.4% ± 2.8% **
483 ± 18 **
513 ± 23 **
106.2%
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Mussels
DTX1
9.9% ± 5.4% **
822 ± 21 **
866 ± 28 **
105.4%
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Clams
DTX1
95.0% ± 0.6% **
540 ± 31 **
556 ± 28 **
103.0%
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Mussels
DTX1
8.3% ± 1.3% **
1382 ± 86 **
1863 ± 176 **
134.8%
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Mussels
DTX1
11.8% ± 3.5% **
1983 ± 201 **
2372 ± 117 **
119.6%
Sample (Source: US)
LC-MS/MS analysis of hydrolysed extracts LC-MS/MS extract LFIA extract (µg (µg OA eq/kg) OA eq/kg)
LFIA recovery
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*McNabb protocol (LC-MS/MS extracts analysed 2008, US; LFIA extracts analysed 2011, UK). ** Mean ± SD (n = 2 extractions per sample). LC-MS/MS standard working range of 25 - 1600 µg/kg
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Table 2. LFIA results in comparison to reference methods Sample (source region) 1 (EU) 2 (EU) 3 (EU) 4 (EU) 5 (EU) 6 (EU) 7 (EU) 8 (EU) 9 (EU) 10 (EU) 11 (EU) 12 (EU) 13 (US) 14 (US) 15 (US) 16 (US) 17 (US) 18 (US) 19 (US) 20 (US) 21 (US) 22 (US) 23 (US) 24 (US) 25 (US) 26 (EU) 27 (US) 28 (US) 29 (US) 30 (US) 31 (EU) 32 (EU) 33 (EU) 34 (EU) 35 (EU) 36 (EU) 37 (EU)
Species
Toxin profile
Mussels Mussels Mussels Mussels Mussels Mussels Mussels Mussels Mussels Mussels Mussels Mussels Mussels Oysters Oysters Oysters Oysters Oysters Oysters Oysters Oysters Oysters Clams Clams Clams Mussels Mussels Mussels Mussels Mussels Mussels Scallops Scallops Scallops Mussels Mussels Mussels
ND OA OA NA OA OA (98%), DTX1 (2%) OA (74%), DTX1 (18%), DTX2 (8%) OA (99%), DTX1 (1%) OA (92%), DTX2 (8%) OA (99%), DTX1 (1%) OA (99%), DTX1 (1%) OA (99%), DTX1 (1%) ND ND OA OA OA ND OA OA OA ND ND DTX1 ND ND DTX1 DTX1 DTX1 DTX1 OA OA OA (51%), DTX2 (49%) ND OA (37%), DTX1 (15%), DTX2 (49%) OA (38%), DTX1 (57%), DTX2 (5%) OA (63%), DTX2 (37%)
LC-MS/MS, unhydrolysed (µg OA eq/kg) < 25 24.5 55 NA 59 96 149 215 320 269 254 276 < 25 < 25 < 25 < 25 < 40 < 25 < 40 < 40 < 40 < 25 < 25 32 < 25 < 25 485 841 1382 > 1600 83 41 60 < 25 151 109 127
LC-MS/MS, hydrolysed (µg OA eq/kg) < 25 81 122 125 170 269 336 386 415 569 569 618 < 25 < 25 190 376 410 < 25 440 450 470 < 25 < 25 515 < 25 < 25 524 944 > 1600 > 1600 132 66 200 < 25 299 168 243
MBA result
LFIA result unhydrolysed
LFIA result hydrolysed
Confirmatory result
NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA
C C C C C NC NC NC NC NC NC NC C C C C C C C C C C C C C C NC NC NC NC C C C C NC NC NC
C C C NC NC NC NC NC NC NC NC NC C C NC NC NC C NC NC NC C C NC C C NC NC NC NC C C NC C NC NC NC
C C C C NC NC NC NC NC NC NC NC C C NC NC NC C NC NC NC C C NC C C NC NC NC NC C C NC C NC NC NC
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Page 29 of 32 38 (EU) Oysters OA (50%), DTX2 (50%) 39 (EU) Mussels OA (82%), DTX1 (18%) 40 (EU) Mussels OA (85%), DTX2 (15%) 41 (EU) Clams OA (70%), DTX1 (13%), DTX2 (17%) 42 (EU) Oysters OA (56%), DTX1 (8%), DTX2 (37%) 43 (EU) Cockles ND 44 (EU) Cockles ND 45 (EU) Clams ND 46 (EU) Oysters ND 47 (EU) Mussels ND 48 (EU) Mussels ND 49 (EU) Mussels OA (91%), DTX1 (2%), DTX2 (7%) 50 (EU) Mussels OA (55%), DTX1 (27%), DTX2 (18%) 51 (EU) Mussels OA (48%), DTX1 (9%), DTX2 (43%) 52 (EU) Mussels OA (47%), DTX1 (10%), DTX2 (43%) 53 (EU) Mussels OA (75%), DTX1 (4%), DTX2 (21%) 54 (EU) Mussels OA (78%), DTX1 (5%), DTX2 (17%) 55 (EU) Mussels OA (72%), DTX1 (3%), DTX2 (25%) 56 (EU) Mussels OA (76%), DTX1 (24%) 57 (EU) Mussels OA (70%), DTX1 (30%) 58 (EU) Mussels NA 59 (EU) Mussels ND 60 (EU) Mussels ND 61 (EU) Mussels OA (36%), DTX1 (64%) 62 (EU) Mussels NA 63 (EU) Mussels NA 64 (EU) Mussels NA 65 (EU) Mussels NA 66 (EU) Mussels NA 67 (EU) Mussels NA 68 (EU) Mussels NA 69 (EU) Mussels ND 70 (EU) Mussels OA 71 (EU) Mussels OA (99%), DTX1 (1%) 72 (EU) Mussels ND NA = no data. ND = not detected. C = compliant. NC = non-compliant.
Journal of Agricultural and Food Chemistry 206 231 63 491 139 < 25 < 25 < 25 < 25 < 25
