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Development and Validation of a Lateral Flow Immunoassay for the Rapid Screening of Okadaic Acid and All Dinophysis Toxins from Shellfish Extracts
... A lateral flow test involves the shellfish extract transported across a reagent zone in which OA, DTX-1, DTX-2 and DTX-3 specific antibodies are combined with colored particles. If a toxin is present, it is captured by the particle-antibody complex, and as its concentration increases, the intensity of the test "line" decreases [19]. ...
... In 2015, Jawaid et al. reported on the development and validation of a new rapid test kit, the Neogen LFA, this time a qualitative test strip/reader for the OA group toxins in shellfish [19]. This validation method tested both spiked (OA, DTX-1, DTX-2 and DTX-3 with hydrolysis procedure) and naturally contaminated shellfish (mussels, scallops, oysters, and clams) and compared the results to LC-MS/MS. ...
... Finally, one sample (~3 g) of the CRM DSP-Mus-c was sent to each laboratory as a positive control. In total, 19 samples (randomly numbered [1][2][3][4][5][6][7][8][9][10][11][12][13][14][15][16][17][18][19] were dispatched frozen to each of four laboratories (Table 11). ...
Rapid methods for the detection of biotoxins in shellfish can assist the seafood industry and safeguard public health. Diarrhetic Shellfish Toxins (DSTs) are produced by species of the dinoflagellate genus Dinophysis, yet the comparative efficacy of their detection methods has not been systematically determined. Here, we examined DSTs in spiked and naturally contaminated shellfish–Sydney Rock Oysters (Saccostrea glomerata), Pacific Oysters (Magallana gigas/Crassostrea gigas), Blue Mussels (Mytilus galloprovincialis) and Pipis (Plebidonax deltoides/Donax deltoides), using LC-MS/MS and LC-MS in 4 laboratories, and 5 rapid test kits (quantitative Enzyme-Linked Immunosorbent Assay (ELISA) and Protein Phosphatase Inhibition Assay (PP2A), and qualitative Lateral Flow Assay (LFA)). We found all toxins in all species could be recovered by all laboratories using LC-MS/MS (Liquid Chromatography—tandem Mass Spectrometry) and LC-MS (Liquid Chromatography—Mass Spectrometry); however, DST recovery at low and mid-level concentrations (<0.1 mg/kg) was variable (0–150%), while recovery at high-level concentrations (>0.86 mg/kg) was higher (60–262%). While no clear differences were observed between shellfish, all kits delivered an unacceptably high level (25–100%) of falsely compliant results for spiked samples. The LFA and the PP2A kits performed satisfactorily for naturally contaminated pipis (0%, 5% falsely compliant, respectively). There were correlations between spiked DSTs and quantitative methods was highest for LC-MS (r² = 0.86) and the PP2A kit (r² = 0.72). Overall, our results do not support the use of any DST rapid test kit as a stand-alone quality assurance measure at this time.
... The same group has produced a similar assay for the assessment of ASP and DSPs [127,128]. The DSP detection kit can detect OA-group toxins in scallops, clams, and mussels [101] as well as oysters [91,100]. ...
... The DSP detection kit can detect OA-group toxins in scallops, clams, and mussels [101] as well as oysters [91,100]. Neogen advertises an LOD of 160 µg/kg, although values as low as 60 µg/kg can test positive [128]. This can be frustrating for producers when their harvest is well below the action limit, but still testing positive by LFIA. ...
... Methanol has been used widely for extraction of regulated lipophilic toxins such as OA, DTXs, AZAs, YTXs and PTXs, as well as other lipophilic toxin groups such as the cyclic imines and palytoxins. Detection of OA-group esters requires an additional alkaline hydrolysis step to account for the added acetyl group [101,117,128]. Alternatively, ethanol has been used to extract OA, DA, and STX for detection by LC-MS/MS and ELISA [81]. ...
In the past twenty years marine biotoxin analysis in routine regulatory monitoring has advanced significantly in Europe (EU) and other regions from the use of the mouse bioassay (MBA) towards the high-end analytical techniques such as high-performance liquid chromatography (HPLC) with tandem mass spectrometry (MS). Previously, acceptance of these advanced methods, in progressing away from the MBA, was hindered by a lack of commercial certified analytical standards for method development and validation. This has now been addressed whereby the availability of a wide range of analytical standards from several companies in the EU, North America and Asia has enhanced the development and validation of methods to the required regulatory standards. However, the cost of the high-end analytical equipment, lengthy procedures and the need for qualified personnel to perform analysis can still be a challenge for routine monitoring laboratories. In developing regions, aquaculture production is increasing and alternative inexpensive Sensitive, Measurable, Accurate and Real-Time (SMART) rapid point-of-site testing (POST) methods suitable for novice end users that can be validated and internationally accepted remain an objective for both regulators and the industry. The range of commercial testing kits on the market for marine toxin analysis remains limited and even more so those meeting the requirements for use in regulatory control. Individual assays include enzyme-linked immunosorbent assays (ELISA) and lateral flow membrane-based immunoassays (LFIA) for EU-regulated toxins, such as okadaic acid (OA) and dinophysistoxins (DTXs), saxitoxin (STX) and its analogues and domoic acid (DA) in the form of three separate tests offering varying costs and benefits for the industry. It can be observed from the literature that not only are developments and improvements ongoing for these assays, but there are also novel assays being developed using upcoming state-of-the-art biosensor technology. This review focuses on both currently available methods and recent advances in innovative methods for marine biotoxin testing and the end-user practicalities that need to be observed. Furthermore, it highlights trends that are influencing assay developments such as multiplexing capabilities and rapid POST, indicating potential detection methods that will shape the future market.
... In a comparison of the performance of four PSP toxins test kits on Tasmanian shellfish, the LFA Neogen test developed by Jawaid et al. (2015) proved to be the most suitable based on sensitivity, ease of use, and performance. The ELISA Abraxis and Europroxima tests underestimated PSTs in 35e100% of the samples. ...
... For Table 4 Performance characteristics of commercially available kits for PSPs analysis of shellfish tissues (Mcleod et al., 2015). (Jawaid et al., 2015). k SLV shows 6/23 naturally contaminated samples gave false positive results when toxin levels were <MPL by LC-FLD or MBA (Jawaid et al., 2015). ...
... (Jawaid et al., 2015). k SLV shows 6/23 naturally contaminated samples gave false positive results when toxin levels were <MPL by LC-FLD or MBA (Jawaid et al., 2015). l Scotia report 1 false negative from 3492 samples. ...
The objective of this critical review was to provide a comprehensive summary of paralytic shellfish toxins (PSTs) producing species and knowledge gaps in detecting PSTs in drinking water resources, with a focus on recent development of PSTs monitoring methods and tools for drinking water monitoring. PSTs, which are also called Saxitoxins (STXs), are a group of neurotoxins not only produced by marine dinoflagellates but also freshwater cyanobacteria. The presence of PSTs in freshwater has been reported from all continents except Antarctica. PSTs in poisoned sea food such as shellfish, molluscs and crustaceans may attack the nerve system after consumption. The high incidences of PSTs occurring in drinking water sources showed another route of potential human exposure. A development of simple and fast screening tools for drinking water surveillance of PSTs is needed. Neurotoxins produced by freshwater cyanobacteria are understudied relative to microcystin and little study is done around PSTs in drinking water monitoring. Some fast screening methods exist. The critical issues for using them in water surveillance, particularly matrix effect and cross-reactivity are summarized, and future research directions are high-lighted. We conclude that monitoring routines at drinking water resources should start from species level, followed by a profound screening of toxin profile. For practical monitoring routine, fast screening methods should be combined with highly sensitive and accurate analytical methods such as liquid chromatography/liquid chromatography–mass spectrometry (LC/LC-MS). A thorough understanding of toxin profile in source water is necessary for screening tool selection.
... Compared with confirmatory instrumental approaches, biosensor and nanotechnology for diagnostics could provide rapid screening results and the availability of specific and onsite detection (Hayat, Barthelmebs, Sassolas, & Marty, 2012;Llamas et al., 2007;Su, Qiu, Fang, Zou, & Wang, 2017;Yakes, Buijs, Elliott, & Campbell, 2016). For example, versatile designed lateral-flow immunoassay (LFIA) technology based on colloidal gold or Fluorescent microspheres provides alternative rapid protocols (Jawaid et al., 2015;. ...
... DTX-1 had an additional methyl group (35-methyl-OA, see Fig. 3S of supplementary materials) and produces an assured colorimetric reaction under the current immunoassay approach. Due to the unavoidable cross-activity effects, it was reported that toxicity equivalency factors (TEFs) were assigned equally for OA and DTX-1 (Jawaid et al., 2015). Despite this existence of crossreactivity, in this study, we should note that the IC 50 of DTX-1 is 8.18 µg L −1 , which is higher than the value of 3.26 µg L −1 of OA, equal to the value of 160 µg kg −1 in a real sample. ...
... Despite this existence of crossreactivity, in this study, we should note that the IC 50 of DTX-1 is 8.18 µg L −1 , which is higher than the value of 3.26 µg L −1 of OA, equal to the value of 160 µg kg −1 in a real sample. Jawaid and coworkers reported that samples containing at or greater than 160 µg kg −1 will generate noncompliant results, and a 95% accuracy level at correctly identifying 60 µg kg −1 samples as compliant was observed in a lateral flow immunoassay study (Jawaid et al., 2015). The results from naturally contaminated samples indicated no false compliant results and no false noncompliant results at <50% MPL (OA is 160 µg kg −1 ), and these results implied that the DTX-1 will not produce obvious interference in the OA analysis, especially at the lower level of OA in real samples. ...
Using newly designed epoxy group activated carbon shell magnetic beads as a goat against mouse antibody (G-Mab) carrier, a competitive immunoassay was conducted on the surface of G-Mab-coated magnetic beads (G-Mab-MBs). Mouse monoclonal antibody against Okadaic acid (MAb), Okadaic acid-horseradish peroxidase conjugate (OA-HRP) and OA were simultaneously added into a reaction system. The enzyme-linked immunosorbent assay (ELISA) was performed in a 96-well microplate incorporated with magnetic bars. Detection of the OA level by using the current approach provided an excellent linear relationship in the range of 0.35–25.0 µg L⁻¹ with a half-maximal inhibitory concentration IC50 of 3.27 µg L⁻¹ and LOD values of 0.35 µg L⁻¹. The shellfish matrix and other algal toxins presented in the sample extraction did not produce obvious interference. As a comparison, tests were conducted via commercial ELISA and liquid chromatography-tandem mass spectrometry (LC-MS/MS), and the corresponding analytical results demonstrated the reliability of the current protocol.
... Only the solutions, consumables, and equipment supplied with the kits were used. DSP-toxin analysis was conducted according to the method described by Jawaid et al. (2015a). Briefly, the test procedure for the DSP analysis (lot no: 9561-56) was as follows: 2 g (±0.1 g) of homogenized mussel sample was mixed with 8 mL of analytical-grade methanol in a plastic extraction bag containing a mesh filter. ...
The mucilage phenomenon observed in the Sea of Marmara in 2021, has raised public concern about seafood safety. Mediterranean mussels serve as a vehicle in food chain, enabling the transfer of pollutants. Farmed and wild mussels were collected from 4 different stations throughout the fishing season. Biotoxins causing amnesic, paralytic, or diarrhetic shellfish poisonings (ASP, PSP, or DSP) were examined during monthly samplings. Potential health risks posed by cadmium, lead and arsenic were assessed. All health risks were evaluated considering 150 g/week mussel consumption considering different age groups of consumers (50, 60, 70 kg). Estimated Weekly Intake calculations of metals were determined to be lower than Provisional Tolerable Weekly Intake at all age groups throughout the sampling period in all stations. Target Hazard QuotientCd of mussels captured from Istanbul Strait was always determined <1, while it was equal to 1 for 50 kg individuals in Gelibolu samples. All THQAs were >1. Target carcinogenic Risk was evaluated for Pb and iAs, which were found to be negligible and acceptable, respectively. No biotoxins responsible for ASP, PSP, or DSP were detected. Hg levels were under detectable limits. Excluding Cd, the results did not reveal any risks associated with mussel consumption during mucilage.
... DTX1 is a member of the human Deltex (DTX) protein family, located on chromosome 12 (12q24.13), and its 67.4 kDa encoded protein contains 620 amino acids (Jawaid et al., 2015). Our research has shown that DTX1 is an important target for HBV inhibition, which could inhibit HBV replication and translation. ...
Background & Aims
Current antiviral drugs, including nucleoside analogs and interferon, fail to eliminate the HBV covalently closed circular DNA (cccDNA), which serves as a transcript template in infected hepatocytes. Silencing the HBV X protein, which plays a crucial role in cccDNA transcription, is a promising approach to inhibit HBV replication. Therefore, the identification of novel compounds that can inhibit HBx-mediated cccDNA transcription is critical.
Methods
Initially, a compound library consisting of 715 monomers derived from traditional Chinese medicines known for their liver-protecting properties was established. Then, MTT assays were used to determine the cytotoxicity of each compound. The effect of candidates on Flag-HBx expression was examined by real-time PCR and western blotting in Flag-HBx transfected HepG2-NTCP cells. Ultimately, the antiviral effect of gambogic acid (GA) on HBV was observed in HBV-infected HepG2-NTCP cells. Mechanistically, the functional role of DTX1 in GA-induced HBV inhibition was examined using RNA-seq. Finally, the antiviral effect of GA was estimated in vivo.
Results
Gambogic acid (GA), a natural bioactive compound with a myriad of biological activities, markedly reduced Flag-HBx expression. Potent and dose-dependent reductions in extracellular HBV RNAs, HBV DNA, HBsAg, HBeAg and HBc protein were discovered three days after GA treatment in HBV-infected cells, accompanied by the absence of significant cytotoxicity. Furthermore, our research revealed that GA exhibited a dose-dependent inhibition of HBx expression, which is a pleiotropic protein required for HBV infection in vivo. We explored the mechanisms underlying GA-mediated inhibition of HBV and confirmed that this inhibition is accomplished by upregulating the expression of the DTX1 gene and boosting the Notch signaling pathway. Finally, the inhibitory effect of GA on HBV replication was tested in vivo using a mouse model of hepatitis B virus recombinant cccDNA.
Conclusions
Herein, we discovered GA, which is a natural bioactive compound that targets HBx to inhibit hepatitis B virus replication by activating the DTX1-Notch signaling pathway.
... ELISA is a sensitive method that allows for testing dozens of samples simultaneously, but it cannot be considered fast because it is carried out in several stages (incubations) with microplate washing after each. A much faster immunoanalytical method is the immunochromatographic analysis (ICA), which allows for obtaining results in 10-15 min with ready-to-use test strips [17][18][19][20]. Among the shortcomings of the ICA, the time required for the assembly of a multi-membrane composite (test strips), including the application of specific immunoreagents on the membrane carriers, drying, cutting, etc., can be noted. ...
In this study, a homogeneous fluorescence polarization immunoassay (FPIA) for the detection of hazardous aquatic toxin okadaic acid (OA) contaminating environmental waters was for the first time developed. A conjugate of the analyte with a fluorophore based on a fluorescein derivative (tracer) was synthesized, and its interaction with specific anti-OA monoclonal antibodies (MAbs) was tested. A MAbs–tracer pair demonstrated highly affine immune binding (KD = 0.8 nM). Under optimal conditions, the limit of OA detection in the FPIA was 0.08 ng/mL (0.1 nM), and the working range of detectable concentrations was 0.4–72.5 ng/mL (0.5–90 nM). The developed FPIA was approbated for the determination of OA in real matrices: river water and seawater samples. No matrix effect of water was observed; therefore, no sample preparation was required before analysis. Due to this factor, the entire analytical procedure took less than 10 min. Using a compact portable fluorescence polarization analyzer enables the on-site testing of water samples. The developed analysis is very fast, easy to operate, and sensitive and can be extended to the determination of other aquatic toxins or low-molecular-weight water or food contaminants.
... OA extraction from certified reference mussels The samples were prepared using a modified version of previously reported methods (Jawaid et al., 2015). We acquired the certified negative mussel samples (CRM-Zero-Mus) from the NRC. ...
Okadaic acid (OA) and the analogs of dinophysistoxin (DTX) are important members of diarrhetic shellfish poisoning (DSP) toxins. In this study, five OA-specific mAbs (monoclonal antibodies) were developed from five stable cells of hybridoma. The anti-OA mAb-2D7 showed high sensitivity to OA, the IC50 of the antibody was 0.24 ng/mL, and its cross-reactivity was 91.6% with DTX-1 and 110.5% with DTX-2., In dcELISA, the IC50 was set at 0.182 ng/mL, and the detection limit was set at 0.023 ng/mL by using the anti-OA mAb-2D7. The level of OA recovered from spiked mussel samples was 2–50 ng/g, ranging from 97.6 ± 7.2% to 106.4 ± 9.8%. In contrast, the immunostrip assay with a limit of 5 ng/mL, conducted for detecting OA, was completed in 10 min. The mAb-based dcELISA and immunostrip assay developed were precise and sensitive enough to quickly assess OA, DTX-1, and DTX-2 in shellfish specimens.
... Lateral flow immunoassays (LFIA) are suitable for unexperienced end-users and have been used for multiple applications. LFIAs have also been adapted to marine toxin detection, including PSP toxins, OA and DTXs, being commercially available for a while even in a dipstick form [361][362][363][364]. In addition, immunoassays are easily adapted to high-throughput screening [365,366]. ...
Marine phycotoxins are a multiplicity of bioactive compounds which are produced by microalgae and bioaccumulate in the marine food web. Phycotoxins affect the ecosystem, pose a threat to human health, and have important economic effects on aquaculture and tourism worldwide. However, human health and food safety have been the primary concerns when considering the impacts of phycotoxins. Phycotoxins toxicity information, often used to set regulatory limits for these toxins in shellfish, lacks traceability of toxicity values highlighting the need for predefined toxicological criteria. Toxicity data together with adequate detection methods for monitoring procedures are crucial to protect human health. However, despite technological advances, there are still methodological uncertainties and high demand for universal phycotoxin detectors. This review focuses on these topics, including uncertainties of climate change, providing an overview of the current information as well as future perspectives.
... Immunoassays, including enzyme-linked immunosorbent assay (ELISA) (Abad et al., 1999;Gui, Jin, Sun, Guo, & Zhu, 2009;Lan et al., 2019;Yao, Liu, Song, Kuang, & Xu, 2017) and immunochromatographic strip assay (Guo, Liu, Gui, & Zhu, 2009;Zhao et al., 2019;Zhou et al., 2004), are time-saving, sensitive, and highthroughput. Colloidal gold-based strip immunoassay provides a portable detection with visual observation in the field Jawaid et al., 2015;Wang et al., 2015;Wang et al., 2017), which requires relatively short development time that brings applications faster to the market. Strip-based immunoassays are specific, sensitive and sample pre-treatment simplified and could obtain the result within 5 min. ...
It is long-cherished to develop rapid approaches for the identification and quantification of pesticide and its metabolites. Carbofuran (CBF) and its metabolite 3-hydroxy-carbofuran (3-OH-CBF) are highly toxic to humans and non-target organisms, which makes it mandatory for the detection of their residues in foods and the environment in many countries. Herein, a colloidal gold-strip assay based on a broad-specific monoclonal antibody (mAb) for simultaneous determination of CBF and 3-OH-CBF was developed. The colloidal gold-strips have a runtime within 5 min without complex sample pretreatment and perform a cut-off limit of detection (LOD) of 7–10 ng/mL for both carbofuran and 3-OH-CBF. The developed portable strip assay is a versatile and robust tool for the rapid and simultaneous detection of CBF and 3-OH-CBF in water samples as well as for the determination of illegal addition of CBF in other pesticide preparations.
... Contrary to OA, DTX1 possesses an additional methyl group, and DTX2 is an isomer of OA. The only difference between OA and two latter compounds is the position of the methyl group (Fig. S1) (Jawaid et al., 2015). As shown in Fig. 4B, although DTX1 and DTX2 share similar structure to that of OA, the FL quenching properties were slightly affected with the increased C DTX1 /C OA and C DTX2 /C OA ratios, which implied efficient imprinting developed onto the MIS-QDs surface. ...
Molecularly imprinted polymers (MIPs) are attracting substantial interest as artificial plastic antibodies because of their biometric capability for targeting small molecules. In this study, molecularly imprinted silica material-coated quantum dots (MIS-QDs) with selective recognition capability to okadaic acid (OA) were developed and characterized. The synthesized MIS-QDs with specific imprinting cavities exhibited excellent recognition capability similar to those of biological antibodies and high fluorescence (FL) quenching selectivity for OA. Furthermore, the MIS-QDs with unsaturated bonds were immobilized onto the surface of 96-well microplates by cold plasma-induced grafting. A novel direct competitive microplate assay strategy was then proposed. The FL quenching properties of the developed microplate assay showed an excellent linear relationship with OA in the range of 10.0-100.0 μg/kg with a correlation coefficient of 0.9961. The limit of detection for OA was 0.25 μg/kg in the shellfish samples. The mean quantitative recoveries were 92.5%-101.0% and 92.9%-101.3%, with relative standard deviations of <7.7% and 7.6% for pure solvents and purified shellfish samples, respectively. The established microplate assay strategy can be used as a rapid and high-throughput method for analyzing OA marine toxins in biological samples.
... Although successfully developed, most of the antibody based PSP toxin methods suffer from cross-reactivity problems with certain PSP toxin profiles. For DSP toxins, LFD devices have been developed and validated (Jawaid et al., 2015). The validation resulted in good results and acceptable cross-reactivity was obtained for the various toxin analogues. ...
Over the past decades, accumulation of phycotoxins (toxins from marine algae) in shellfish and other marine biotoxins in seafood has received increasing attention. The latter is largely due to the greater number of algal blooms, the poisoning symptoms in humans resulting from ‘new’ biotoxins in areas where they had until recently not been observed and, finally, the discovery of new, highly potent toxins. The most frequently detected marine biotoxins represent a chemically very heterogeneous group of phycotoxins, the toxicity of which is generally related to a specific interaction of these toxins with ion channels of excitable membranes. Besides these neurotoxins, inhibitors of protein phosphatases have been described. On the basis of the clinical symptoms observed, we distinguish two groups of marine biotoxins: (1) diarrhetic shellfish poisoning toxins; and (2) marine neurotoxic biotoxins that can be found in seafood. In addition, primarily prevalent in fresh water, toxins can be synthesized by cyanobacteria that are also classified as algal toxins. These toxins, which exert hepatotoxic or neurotoxic effects, are mainly relevant as potential contaminants of drinking water and food supplements and are not included in this chapter.
... A number of LFIC/LFIA/LFA-based assays for some of the major algal toxin groups (i.e., DSP, ASP, and PSP) have been developed, and several of these are commercially available, primarily through either Scotia Rapid Testing Ltd. (formerly Jellett Rapid Testing Ltd.) or Neogen Europe Ltd. The PSP toxin assay manufactured by Scotia Rapid Testing was adopted by the U.S. ISSC as an "Approved Limited Use Method for Marine Bio toxin Testing" (i.e., it can be used to determine when to perform a MBA in a previously closed area, a negative result can be substituted for a MBA to maintain an area in the open status, and a positive result shall be used for a precautionary closure; note that this status refers only to use with the AOAC Mouse Bioassay [OMA 959.08] extraction protocol) for use in the NSSP (2013), whereas the Neogen DSP, ASP, and PSP toxin assays have undergone single-laboratory validation (Jawaid et al., 2013(Jawaid et al., , 2015a(Jawaid et al., , 2015b. In addition, other investigators have conducted independent evaluations of certain products with results reported in the peer-reviewed literature (e.g., Vale et al., 2009;Laycock et al., 2010;Eberhart et al., 2013;DeGrasse et al., 2014;Turner et al., 2015aTurner et al., , 2015bJohnson et al., 2016). ...
The ability to mitigate the adverse impacts of harmful algal blooms (HAB) on humans, wildlife, fisheries, and ecosystems, as well as to identify the environmental factors driving HAB population growth and toxicity, is based largely on early detection of causative organisms and their toxins. This chapter explores a wide range of organism and toxin detection methods and technologies. It describes the fundamental principles of operation/conduct. It outlines the prospects for future advances in bloom and toxin detection/surveillance capabilities. Molecular methods used to detect organisms are potentially faster and more accurate than light microscopy (LM) methods. Monoclonal antibodies (MAbs) and polyclonal antibodies have been used to detect cultured and field‐collected cells of a wide variety of harmful algae. Immunoassays are based on antibodies recognizing and binding to one or more epitopes or antigenic determinants on a toxin molecule. The Enzyme‐Linked Immunosorbent Assay, ELISA has been the format of choice for most commercial algal toxin detection kits.
... Immunoassays, including the enzyme-linked immunosorbent assay (ELISA), immunochromatographic assay (ICA), immunochip and other immunosensors as simple and sensitive methods, are more suitable for rapid and high-throughput analysis of multiple compounds. (Jawaid et al., 2015;Jiang et al., 2013;Li et al., 2011;, Song et al., 2014Song et al., 2015). Among these assays, the immunochromatographic assay is the easiest to use, time-saving and cost-effective. ...
Thiacloprid (TCL) is a neonicotinoid insecticide. Its widespread use has led to high levels of residue in fruits and vegetables. Hence, it is important to detect TCL rapidly, accurately, and sensitively in fruits and vegetables. Recombinant antibodies (rAbs) can be synthesized rapidly with little batch-to-batch variation. In this study, recombinant single-chain variable fragment (scFv) antibody and full-length recombinant antibody against TCL were produced using three different expression systems (E. coli, yeast, and mammalian cell). The results of SDS-PAGE and non - competitive enzyme-linked immunosorbent assay (ELISA) indicated that the full-length rAb exhibited promising characteristics, and the IC50 value of indirect competitive ELISA (ic-ELISA) was 2.63 μg L-1. However, recombinant scFv antibody had little affinity for the antigen. To understand antibody recognition, the three-dimensional (3D) model of the variable fragment (Fv) was built via homologous modeling. The interaction between Fv and TCL was analyzed via molecular docking and the results of molecular docking showed that VAL-158, ALA-211, PHE-220, TRP-218, TRP-49, and ILE-100 were mainly responsible for antibody recognition. In addition, a time-resolved fluorescent microsphere-immunochromatographic test strip (TRFM-ICTS) was developed with a linear range and limit of detection of 0.01-10 ng mL-1 and 0.003 ng mL-1 within 15 min under optimal conditions. The IC50 value was 4.268 ng mL-1, and the recovery ranged between 79.4% and 118.6%, which was consistent with HPLC-MS. The TRFM-ICTS has great advantages in sensitivity and applicability.
A multiplex lateral flow immunoassay (LFA) has been developed to detect the primary marine biotoxin groups: amnesic shellfish poisoning toxins, paralytic shellfish poisoning toxins, and diarrhetic shellfish poisoning toxins. The performance characteristics of the multiplex LFA were evaluated for its suitability as a screening method for the detection of toxins in shellfish. The marine toxin-specific antibodies were class-specific, and there was no cross-reactivity between the three toxin groups. The test is capable of detecting all three marine toxin groups, with working ranges of 0.2-1.5, 2.5-65.0, and 8.2-140.3 ng/mL for okadaic acid, saxitoxin, and domoic acid, respectively. This allows the multiplex LFA to detect all three toxin groups at the EU regulatory limits, with a single sample extraction method and dilution volume. No matrix effects were observed on the performance of the LFA with mussel samples spiked with toxins. The developed LFA uses a simple and pocket-sized, portable Cube Reader to provide an accurate result. We also evaluated the use of this Cube Reader with commercially available monoplex lateral flow assays for marine toxins.
The pollution caused by estrogens in the environment and food has received increasing attention. It is still challenging for on-site immunochromatographic assay (ICA) detection of estrogens. The performance of the prepared probes plays a decisive role in the sensitivity and stability of the ICA system. The published probes usually directly couple the detection antibody to the label, ignoring the influence of the label on the activity of the antibody. In this study, 17β-estradiol (E2) was used as a model analyte for the ICA system. Two universal probes were constructed based on quantum dot nanobeads (QBs), recombinant protein A (SPA, from Staphylococcus aureus), and rabbit anti-mouse immunoglobulin G antibody (anti-IgG). The probes were prepared by coupling QBs with SPA, releasing anti-E2 monoclonal antibody (mAb), and maintaining its activity. The prepared universal probes can orient recognize the Fc region of mAb and fully expose its Fab region, improving the detection sensitivity of the ICA system. The free anti-E2 mAb and the universal probe ([email protected] or [email protected]@anti-IgG) were used as the detection antibodies and signal donors, respectively.
The results show that the proposed ICA based on [email protected] and [email protected]@anti-IgG probes could detect E2 with IC50 of 8.83 and 0.93 ng/mL, respectively, within 15 min under optimal conditions. The recovery results of ICA based on [email protected] and [email protected]@anti-IgG probes showed good agreement with the findings of the high-performance liquid chromatography (HPLC) analysis for spiked samples. The developed ICA system based on universal probes was superior in terms of sensitivity, rapidity, and applicability, and held great promise for its implementation in detecting environmental and food small-molecule pollutants.
Phallotoxins, toxic cyclopeptides found in wild poisonous mushrooms, are predominant causes of fatal food poisoning. For the early and rapid diagnosis mushroom toxin poisoning, a highly sensitive and robust monoclonal antibody (mAb) against phallotoxins was produced for the first time. The half-maximum inhibition concentration (IC50) values of the mAb-based indirect competitive ELISAs for phallacidin (PCD) and phalloidin (PHD) detection were 0.31 ng mL−1 and 0.35 ng mL−1, respectively. In response to the demand for rapid screening of the type of poisoning and accurate determination of the severity of poisoning, colloidal gold nanoparticle (GNP) and time-resolved fluorescent nanosphere (TRFN) based lateral flow assays (LFA) were developed. The GNP-LFA has a visual cut-off value of 3.0 ng mL−1 for phallotoxins in human urine sample. The TRFN-LFA provides a quantitative readout signal with detection limit of 0.1 ng mL−1 in human urine sample. In this study, urine samples without pretreatment were used directly for the LFA strip tests, and both two LFAs were able to accomplish analysis within 10 min. The results demonstrated that LFAs based on the newly produced, highly sensitive, and robust mAb were able to be used for both rapid qualitative screening of the type of poisoning and accurate quantitative determination of the severity of poisoning after accidental ingestion by patients of toxic mushrooms.Graphical abstract
The risk for seafood poisoning has grown exponentially over the past 50 years will likely continue to increase over the next decade. The most common cause of a seafood-related illness is scombroid poisoning; however, poisonings associated with harmful algal bloom (HAB)-related toxins continue to rise. These algal toxins include ciguatoxins, saxitoxins, domoic acid, brevetoxins, okadaic acid, azaspiracids, and tetrodotoxins, and the majority are neurotoxic. Symptoms vary from mild to severe and fatalities have been reported in some cases. Since there is no antidote for these illnesses, treatment is largely symptomatic. Rapid and accurate diagnosis would be facilitated by advanced studies of exposure biomarkers as well as human thresholds for illness. In the United States, aggressive monitoring of endemic coastal regions is under way with risk communication serving as a critical component of illness prevention.
Highly proliferating, tiny phytoplanktons play a basic role in the stabilization of ecosystem, by contributing to photosynthesis. However, some of these are reportedly harmful to other living organisms, including marine life and humans as well. These harmful phytoplanktons are further classified on the basis of the source of production and toxicity. Exposure to the toxins, which are produced by the harmful phytoplanktons, causes lethal intoxication in humans, which ultimately leads to death. The adverse effects to health and socioeconomic conditions rendered by these marine toxins make it crucial to on‐site detection of these before they can reach the market. Effective detective and screening systems are required to eliminate the hazards. In this chapter, the extensive effects and types of marine toxins along with various emerging techniques utilized for biosensing these toxins are discussed.
Rapid, sensitive, point-of-care detection of bacteria is extremely important in food safety. To address this requirement, we developed a new surface-enhanced Raman scattering (SERS)-based lateral flow (LF) strip biosensor combined with recombinase polymerase amplification (RPA) for simultaneous detection of Listeria monocytogenes and Salmonella Enterica Serotype Enteritidis. AuMBA@Ag core-shell nanoparticles were used in this SERS-LF. Highly sensitive quantitative detection is achieved by measuring the characteristic peak intensities of SERS tags. Under optimal conditions, the SERS intensities of MBA at 1077 cm-1 on test lines are used to measure S. Enteritidis (y=1980.6x-539.3, R2=0.9834) and L. monocytogenes (y=1696.0x -844, R2=0.9889), respectively. The limit of detection is 27 CFU/mL for S. Enteritidis and 19 CFU/mL for L. monocytogenes. Significantly, this SERS-LF has high specificity and applicability in the detection of L. monocytogenes and S. Enteritidis in food samples. Therefore, the SERS-LF is a feasible method for the rapid and quantitative detection of a broad range of bacterial pathogens in real food samples.
The detecting labels used for lateral flow immunoassays (LFAs) have been traditionally gold nanoparticles (GNPs) and more recently, luminescent nanoparticles such as quantum dots (QDs). However, these labels have low sensitivity and are costly, in particular for trace detection of mycotoxins in cereals. Here, we provided a simple preparation procedure for amorphous carbon nanoparticles (ACNPs) and described multiplex LFAs employing ACNPs as labels (ACNP-LFAs) for detecting three Fusarium mycotoxins. The analytical performance of ACNPs in LFA was compared with GNPs and QDs using the same immunoreagents, except for the labels, allowing their analytical characteristics to be objectively compared. The visual limit of detection (vLOD) for ACNP-LFAs in buffer was 8-fold better than GNPs and 2-fold better than QDs. Under optimized conditions, the quantitative limit of detection (qLOD) of ACNP-LFAs in maize were as low as 20 μg/kg for deoxynivalenol, 13 μg/kg for T-2 toxin and 1 μg/kg for zearalenone. These measurements were much lower than the action level of these mycotoxins in maize. The accuracy and precision of the ACNP-LFAs were evaluated by analysis of spiked and incurred maize samples with recoveries of 84.6-109% and coefficients of variation below 13%. The results of ACNP-LFAs using naturally incurred maize samples showed good agreement with results from HPLC-MS/MS, indicating that ACNPs were more sensitive labels than, and a promising alternative to, GNPs used in LFAs for detecting mycotoxins in cereals.
Marine biotoxins are natural toxins produced by specific algae species. These toxins can accumulate in seafood such as mussels, oysters and fish. Consumption of contaminated seafood may lead to severe intoxication such as memory loss, paralysis, diarrhoea and even death. In order to protect consumers, a wide variety of methods have been developed. Historically, methods were based on in vivo animal assays. Alternatives for these animal unfriendly and unethical assays have been developed and are still under development. Other alternative methods are based on in vitro cell assays, receptor binding assays, immunological assays and analytical chemical techniques. This chapter gives an overview on the available methodologies and their application for the determination of marine biotoxins in seafood.
Diarrhetic shellfish poisoning (DSP) refers to a gastrointestinal disease following the ingestion of bivalve shellfish containing dinoflagellate toxins of lipophilic nature collectively referred to as okadaic acids (OAs). OAs strongly inhibit protein phosphatase 2A (PP2A) and thus are quantifiable by measuring the extent of the enzyme inhibition. We recently produced the recombinant catalytic subunit of human PP2A (rhPP2Ac) by genetic engineering techniques using the baculovirus expression system with High Five insect cells. In this study, we evaluated the suitability of rhPP2Ac for use in a microplate OA assay. The limits of detection and quantitation for OA in the whole meat of the scallop Patinopecten yessoensis were 0.0262 mg/kg and 0.0470 mg/kg respectively, which are well below the regulation level in Japan (0.16 mg/kg whole meat). Differences in lipid content of the scallop did not affect the measurement result. Our results confirm that the assay kit using rPP2Ac is an excellent tool for detecting and quantifying OAs in shellfish.
Biotechnology embraces various physical and chemical phenomenon towards advancement of health diagnostics. Towards such advancement, detection of toxins play an important role. Toxins produce severe health impacts on consumption with high mortality associated in acute cases. The most prominent route of infection and intoxication is through food matrices. Therefore, rapid detection of toxins at low concentrations is the need of modern diagnostics. Lateral flow immunoassays are one of the emergent and popularly used rapid detection technology developed for detecting various kinds of analytes. This review thus focuses on recent advancements in lateral flow immunoassays for detecting different toxins in agricultural food. Appropriate emphasis was given on how the labels, recognition elements or detection strategy has laid an impact on improvement in immunochromatographic assays for toxins. The paper also discusses the gradual change in sensitivities and specificities of assays in accordance with the method of food processing used. The review concludes the major challenges faced by this technology and provides an outlook and insight of ideas to improve it in future.
Keywords: Diagnostics, Toxins, Immunoassay, L.O.D., Commercial LFIA
The illness of three people due to diarrhetic shellfish poisoning (DSP) following their ingestion of recreationally harvested mussels from Sequim Bay State Park in the summer of 2011, resulted in intensified monitoring for diarrhetic shellfish toxins (DSTs) in Washington State. Rapid testing at remote sites was proposed as a means to provide early warning of DST events in order to protect human health and allow growers to test “pre-harvest” shellfish samples, thereby preventing harvest of toxic product that would later be destroyed or recalled. Tissue homogenates from several shellfish species collected from two sites in Sequim Bay, WA in the summer 2012, as well as other sites throughout Puget Sound, were analyzed using three rapid screening methods: a lateral flow antibody-based test strip (Jellett Rapid Test), an enzyme-linked immunosorbent assay (ELISA) and a protein phosphatase 2A inhibition assay (PP2A). The results were compared to the standard regulatory method of liquid chromatography coupled with tandem mass spectroscopy (LC-MS/MS). The Jellett Rapid Test for DSP gave an unacceptable number of false negatives due to incomplete extraction of DSTs using the manufacturer’s recommended method while the ELISA antibody had low cross-reactivity with dinophysistoxin-1, the major toxin isomer in shellfish from the region. The PP2A test showed the greatest promise as a screening tool for Washington State shellfish harvesters.
Diarrhetic shellfish poisoning is a gastrointestinal illness caused by consumption of bivalves contaminated with dinophysistoxins. We report an illness cluster in the United States in which toxins were confirmed in shellfish from a commercial harvest area, leading to product recall. Ongoing surveillance is needed to prevent similar illness outbreaks.
The illness of three people in 2011 after their ingestion of mussels collected from Sequim Bay State Park, Washington State, USA, demonstrated the need to monitor diarrhetic shellfish toxins (DSTs) in Washington State for the protection of human health. Following these cases of diarrhetic shellfish poisoning, monitoring for DSTs in Washington State became formalized in 2012, guided by routine monitoring of Dinophysis species by the SoundToxins program in Puget Sound and the Olympic Region Harmful Algal Bloom (ORHAB) partnership on the outer Washington State coast. Here we show that the DSTs at concentrations above the guidance level of 16 μg okadaic acid (OA) + dinophysistoxins (DTXs)/100 g shellfish tissue were widespread in sentinel mussels throughout Puget Sound in summer 2012 and included harvest closures of California mussel, varnish clam, manila clam and Pacific oyster. Concentrations of toxins in Pacific oyster and manila clam were often at least half those measured in blue mussels at the same site. The primary toxin isomer in shellfish and plankton samples was dinophysistoxin-1 (DTX-1) with D. acuminata as the primary Dinophysis species. Other lipophilic toxins in shellfish were pectenotoxin-2 (PTX-2) and yessotoxin (YTX) with azaspiracid-2 (AZA-2) also measured in phytoplankton samples. Okadaic acid, azaspiracid-1 (AZA-1) and azaspiracid-3 (AZA-3) were all below the levels of detection by liquid chromatography tandem mass spectrometry (LC-MS/MS). A shellfish closure at Ruby Beach, Washington, was the first ever noted on the Washington State Pacific coast due to DSTs. The greater than average Fraser River flow during the summers of 2011 and 2012 may have provided an environment conducive to dinoflagellates and played a role in the prevalence of toxigenic Dinophysis in Puget Sound.
Foodborne outbreaks attributed to marine biotoxins were first reported in Portugal in 1946. A regular monitoring programme was implemented in 1986 for PSP, in 1987 for DSP and in 1996 for ASP. The gradual introduction of HPLC methodologies for DSP and PSP allowed a better understanding of toxin biotransformation by bivalves, supplying more selective and sensitive data than mouse bioassays. A comprehensive exposure assessment from DSP toxins in bivalves from the whole coast was only obtained more recently with the introduction of LC–MS methodology. Data on maximum toxin levels found, geographic distribution, seasonality of toxin families, and frequency of samples above current regulatory limits is presented in order to review the data available on exposure assessment after two decades of monitoring. Contamination with DSP toxins was more severe in estuarine and offshore bivalves from the NW and in offshore Donax spp. from the SW and south coasts. DSP toxins were recurrent every year mainly between late spring/early autumn. PSP toxins appeared intermittently in some years between 1986 and 2006, predominantly in autumn. Bivalves from the entire coast were severely contaminated, although bivalves from the NW coast were affected more often. ASP toxins appeared between spring and autumn around the entire coast, but toxin levels rarely exceeded the regulatory limit. Azaspiracids occurred in trace levels below the regulatory limit. Yessotoxins and pectenotoxins occurred in bivalves but have no known effects on the consumers. Several intoxication outbreaks attributed to PSP and DSP occurred during the two decades of the monitoring programme.
The potential for poisoning of humans through their consumption of shellfish which have themselves consumed biotoxin producing marine phytoplankton exists in the UK. Toxins are bio-accumulated within the shellfish flesh allowing them to reach harmful concentrations. This threat is in most part mitigated by monitoring programmes that assess both the presence of potentially harmful phytoplankton and shellfish flesh toxicity. However, the medical profession in the UK remains relatively ignorant of the potential for biotoxin derived shellfish toxicity, preventing quantification of magnitude, frequency, and severity of health effects in the community or the medical significance of more recently discovered toxins. While the current causative species and their toxins are relatively well characterised there remains a lack of understanding of the factors governing the temporal and spatial appearance of harmful phytoplankton. Expansion of shellfish aquaculture is likely both worldwide and in the UK. Better understanding of how harmful phytoplankton interact with their environment to promote the sporadic harmful blooms that we observe is required to underpin risk assessments.
Marine dinoflagellates of the genus Dinophysis can produce toxins of the okadaic acid (OA) and pectenotoxin (PTX) groups. These lipophilic toxins accumulate in filter-feeding shellfish and cause an illness in consumers called diarrhetic shellfish poisoning (DSP). In 2008, a bloom of Dinophysis led to the closure of shellfish harvesting areas along the Texas coast, one of the first DSP-related closures in the U.S. This event resulted in a broad study of toxin production in isolates of Dinophysis spp. from U.S. waters. In the present study, we compared toxin profiles in geographical isolates of Dinophysis collected in the U.S. (Eel Pond, Woods Hole MA; Martha's Vineyard, MA; and Port Aransas Bay, Texas), and in those from Canada (Blacks Harbour, Bay of Fundy) and Chile (Reloncavi Estuary), when cultured in the laboratory under the same conditions. For each isolate, the mitochondrial cox1 gene was sequenced to assist in species identification. Strains from the northeastern U.S. and Canada were all assigned to Dinophysis acuminata, while those from Chile and Texas were most likely within the D. acuminata complex whereas precise species designation could not be made with this marker. Toxins were detected in all Dinophysis isolates and each isolate had a different profile. Toxin profiles of isolates from Eel Pond, Martha's Vineyard, and Bay of Fundy were most similar, in that they all contained OA, DTX1, and PTX2. The Eel Pond isolate also contained OA-D8 and DTX1-D7, and low levels (unconfirmed structurally) of DTX1-D8 and DTX1-D9. D. acuminata from Martha's Vineyard produced DTX1-D7, along with OA, DTX1, and PTX2, as identified in both the cells and the culture medium. D. acuminata from the Bay of Fundy produced DTX1 and PTX2, as found in both cells and culture medium, while only trace amounts of OA were detected in the medium. The Dinophysis strain from Texas only produced OA, and the one from Chile only PTX2, as confirmed in both cells and culture medium.
A liquid chromatography tandem mass spectrometry (LC-MS/MS) method for the quantitative analysis of lipophilic marine toxins in shellfish extracts (mussel, oyster, cockle and clam) was validated in-house using European Union (EU) Commission Decision 2002/657/EC as a guideline. The validation included the toxins okadaic acid (OA), yessotoxin (YTX), azaspiracid-1 (AZA1), pectenotoxin-2 (PTX2) and 13-desmethyl spirolide-C (SPX1). Validation was performed at 0.5, 1 and 1.5 times the current EU permitted levels, which are 160 µg kg-1 for OA, AZA1 and PTX2 and 1,000 µg kg-1 for YTX. For SPX1, 400 µg kg-1 was chosen as the target level as no legislation has been established yet for this compound. The method was validated for determination in crude methanolic shellfish extracts and for extracts purified by solid-phase extraction (SPE). Extracts were also subjected to hydrolysis conditions to determine the performance of the method for OA and dinophysistoxin esters. The toxins were quantified against a set of matrix-matched standards instead of standard solutions in methanol. To save valuable standard, methanolic extract instead of the homogenate was spiked with the toxin standard. This was justified by the fact that the extraction efficiency is high for all relevant toxins (above 90%). The method performed very well with respect to accuracy, intraday precision (repeatability), interday precision (within-laboratory reproducibility), linearity, decision limit, specificity and ruggedness. At the permitted level the accuracy ranged from 102 to 111%, the repeatability from 2.6 to 6.7% and the reproducibility from 4.7 to 14.2% in crude methanolic extracts. The crude extracts performed less satisfactorily with respect to the linearity (less than 0.990) and the change in LC-MS/MS sensitivity during the series (more than 25%). SPE purification resulted in greatly improved linearity and signal stability during the series. Recently the European Food Safety Authority (EFSA) has suggested that to not exceed the acute reference dose the levels should be below 45 µg kg-1 OA equivalents and 30 µg kg-1 AZA1 equivalents. A single-day validation was successfully conducted at these levels. If the regulatory levels are lowered towards the EFSA suggested values, the official methods prescribed in legislation (mouse and rat bioassay) will no longer be sensitive enough. The validated LC-MS/MS method presented has the potential to replace these animal tests.
Electronic supplementary material
The online version of this article (doi:10.1007/s00216-010-3886-2) contains supplementary material, which is available to authorized users.
A new rapid assay for the okadaic acid group of toxins, based on lateral flow immunochromatographic (LFIC) test strips developed by Jellett Rapid Testing Ltd., was assessed on naturally contaminated bivalves from the Portuguese coast. One prototype was evaluated using samples harvested during 2005, extracted with 80% methanol, followed by dilution with the running buffer of a methanolic extract after alkaline hydrolysis for esters. The second prototype was assessed using samples harvested during 2006, extracted with 100% methanol and, after alkaline hydrolysis, the methanol was evaporated by a nitrogen stream followed by re-suspension with the running buffer. The first prototype failed to detect 20% of samples that were positive by LC-MS in the range 160-480 microg kg(-1), and were classified as negative or trace level by LFIC. The presence of methanol in the extracts made correct detection of toxins more difficult. The second prototype classified as positive all samples above 160 microg kg(-1), as confirmed by LC-MS. However, in the second prototype, matrix effects were a major drawback and led to 45% false positives, particularly for mussels, due to compounds in shellfish extracts interfering with the antibodies and reducing the test line intensity. Extraction with a higher percentage of methanol was thought responsible for these matrix effects. Regarding sample migration, both prototypes needed one hour before reading. In an attempt to speed-up sample preparation, a direct digestion of bivalve tissues with sodium hydroxide was evaluated. Low recoveries for esters were found by LC-MS with this hydrolysis technique compared to conventional hydrolysis of methanolic extracts. While prototype A was not sensitive enough, prototype B had too many false positives to be of use to the shellfish industry or in a monitoring program.
Diarrheic shellfish poisoning (DSP) is a recurrent gastrointestinal illness in Morocco, resulting from consumption of contaminated shellfish. In order to develop a rapid and reliable technique for toxins detection, we have compared the results obtained by a commercial immunoassay-“DSP-Check” kit” with those obtained by LC-MS. Both techniques are capable of detecting the toxins in the whole flesh extract which was subjected to prior alkaline hydrolysis in order to detect simultaneously the esterified and non esterified toxin forms. The LC-MS method was found to be able to detect a high level of okadaic acid (OA), low level of dinophysistoxin-2 (DTX2), and surprisingly, traces of azaspiracids 2 (AZA2) in mussels. This is the first report of a survey carried out for azaspiracid (AZP) contamination of shellfish harvested in the coastal areas of Morocco. The “DSP-Check” kit was found to detect quantitatively DSP toxins in all contaminated samples containing only OA, provided that the parent toxins were within the range of detection and was not in an ester form. A good correlation was observed between the two methods when appropriate dilutions were performed. The immunoassay kit appeared to be more sensitive, specific and faster than LC-MS for determination of DSP in total shellfish extract.
A method that uses liquid chromatography with tandem mass spectrometry (LC/MS/MS) has been developed for the highly sensitive and specific determination of amnesic shellfish poisoning toxins, diarrhetic shellfish poisoning toxins, and other lipophilic algal toxins and metabolites in shellfish. The method was subjected to a full single-laboratory validation and a limited interlaboratory study. Tissue homogenates are blended with methanol-water (9 + 1), and the centrifuged extract is cleaned up with a hexane wash. LC/MS/MS (triple quadrupole) is used for quantitative analysis with reversed-phase gradient elution (acidic buffer), electrospray ionization (positive and negative ion switching), and multiple-reaction monitoring. Ester forms of dinophysis toxins are detected as the parent toxins after hydrolysis of the methanolic extract. The method is quantitative for 6 key toxins when reference standards are available: azaspiracid-1 (AZA1), domoic acid (DA), gymnodimine (GYM), okadaic acid (OA), pectenotoxin-2 (PTX2), and yessotoxin (YTX). Relative response factors are used to estimate the concentrations of other toxins: azaspiracid-2 and -3 (AZA2 and AZA3), dinophysis toxin-1 and -2 (DTX1 and DTX2), other pectenotoxins (PTX1, PTX6, and PTX11), pectenotoxin secoacid metabolites (PTX2-SA and PTX11-SA) and their 7-epimers, spirolides, and homoYTX and YTX metabolites (45-OHYTX and carboxyYTX). Validation data have been gathered for Greenshell mussel, Pacific oyster, cockle, and scallop roe via fortification and natural contamination. For the 6 key toxins at fortification levels of 0.05-0.20 mg/kg, recoveries were 71-99% and single laboratory reproducibilities, relative standard deviations (RSDs), were 10-24%. Limits of detection were <0.02 mg/kg. Extractability data were also obtained for several toxins by using successive extractions of naturally contaminated mussel samples. A preliminary interlaboratory study was conducted with a set of toxin standards and 4 mussel extracts. The data sets from 8 laboratories for the 6 key toxins plus DTX1 and DTX2 gave within-laboratories repeatability (RSD(R)) of 8-12%, except for PTX-2. Between-laboratories reproducibility (RSDR) values were compared with the Horwitz criterion and ranged from good to adequate for 7 key toxins (HorRat values of 0.8-2.0).
A series of food poisonings resulting from eating mussels and scallops harvested in the Tohoku District occurred in late June of both 1976 and 1977. A total of 164 persons were officially documented to have suffiered severe vomiting and diarrhea. Toxicity studies using mouse assay on the left-over foods and on shellfishes from the areas where the causative specimens were harvested revealed the presence of an unknown fat-soluble toxin in the hepatopancreas of the shellfishes.
The dominant symptoms in human cases were diarrhea, nausea, vomiting, and abdominal pain. The onset time of these symptoms ranged from 30 minutes to a few hours after ingestion and the victims recovered after three days without any ill effects. Epidemiological data indicated that as little as 12 MU of toxin was enough to induce a mild form of poisoning in humans. In 1976 this remarkable toxicity of shellfishes was recognized only in the areas around Motoyoshi-cho, Miyagi Prefecture. In 1977, however, the toxic region spread along a 100 km stretch of Sanriku Coast from Onagawa Bay, Miyagi Prefecture, to Okirai Bay, Iwate Prefecture. In addition, shellfishes in Mutsu Bay, Aomori Prefecture and in Funka Bay, Hokkaido, also bore the toxin.
The major pectenotoxin and okadaic acid group toxins in Dinophysis acuta and Dinophysis acuminata cell concentrates, collected from various locations around the coast of the South Island of New Zealand (NZ), were determined by liquid chromatography–tandem mass spectrometry (LC–MS/MS). PTX2 and PTX11 were the major polyether toxins in all Dinophysis spp. cell concentrates. D. acuta contained PTX11 and PTX2 at concentrations of 4.7–64.6 and 32.5–107.5 pg per cell, respectively. The amounts of PTX11 and PTX2 in D. acuminata were much lower at 0.4–2.1 and 2.4–25.8 pg per cell, respectively. PTX seco acids comprised only 4% of the total PTX content of both D. acuta and D. acuminata. D. acuta contained low levels of OA (0.8–2.7 pg per cell) but specimens from the South Island west coast also contained up to 10 times higher levels of OA esters (7.0–10.2 pg per cell). Esterified forms of OA were not observed in D. acuta specimens from the Marlborough Sounds. D. acuta did not contain any DTX1 though all D. acuminata specimens contained DTX1 at levels of 0.1–2.4 pg per cell. DTX2 was not present in any New Zealand Dinophysis spp. specimens. Although the total toxin content varied spatially and temporally, the relative proportions of the various toxins in different specimens from the same location appeared to be relatively stable. The total PTX/total OA ratios in different isolates of D. acuta were very similar (mean±S.E.: 14.9±1.9), although the Marlborough Sounds D. acuminata isolates had a higher total PTX/total OA ratio (mean±S.E.: 22.7±2.4) than the Akaroa Harbour isolates (8.0). No evidence of azaspiracids were detected in these specimens. These results show that the LC–MS/MS monitoring of plankton for PTX group toxins (e.g. PTX2) and their derivatives (e.g. PTX2 seco acid) may provide a sensitive, semi-quantitative, indicator of the presence of more cryptic OA group toxins (e.g. OA esters).
Chemistry of marine toxins implicated in seafood poisoning is reviewed. Chemical structure and mode of action are described. Abundant bibliographical information is also provided.
Okadaic acid (OA) and structurally related toxins dinophysistoxin-1 (DTX-1), and DTX-2, are lipophilic marine biotoxins. The current reference method for the analysis of these toxins is the mouse bioassay (MBA). This method is under increasing criticism both from an ethical point of view and because of its limited sensitivity and specificity. Alternative replacement methods must be rapid, robust, cost effective, specific and sensitive. Although published immuno-based detection techniques have good sensitivities, they are restricted in their use because of their inability to: (i) detect all of the OA toxins that contribute to contamination; and (ii) factor in the relative toxicities of each contaminant. Monoclonal antibodies (MAbs) were produced to OA and an automated biosensor screening assay developed and compared with ELISA techniques. The screening assay was designed to increase the probability of identifying a MAb capable of detecting all OA toxins. The result was the generation of a unique MAb which not only cross-reacted with both DTX-1 and DTX-2 but had a cross-reactivity profile in buffer that reflected exactly the intrinsic toxic potency of the OA group of toxins. Preliminary matrix studies reflected these results. This antibody is an excellent candidate for the development of a range of functional immunochemical-based detection assays for this group of toxins.
Jellett Rapid Testing Ltd. has developed a rapid field test kit to screen for diarrhetic shellfish poisoning (DSP) toxins. The new test provides a qualitative (positive/negative) indication of the presence of okadaic acid (OA) and some of its analogues in about 30 min. It is designed as a screening method for regulatory labs to eliminate negative samples, thereby leaving a smaller number of positive samples to be tested with more sophisticated and time-consuming quantitative methods. Due to its simplicity and speed, the Rapid Test for DSP may eventually be used in other applications such as shellfish harvest management and toxin research. The test is based on easy-to-use lateral flow immunochromatographic (LFI) test strips, which operate the same way as Jellett Rapid Testing's Rapid Tests for paralytic shellfish poisoning (PSP) toxins and amnesic shellfish poisoning (ASP) toxins. The sensitivity of the antibodies to some of the analogues of the DSP family of toxins was investigated using pure compounds from the National Research Council of Canada. In the Rapid Test format, okadaic acid, dinophysistoxin 1 (DTX1) and dinophysistoxin 2 (DTX2) were detected similarly with 50% reduction in test line color intensity at 5 nM for the solutions applied to test strips. One of the DTX-3 esters eliminated the test line at 500 nM, indicating low cross-reactivity, whereas no effect was observed with one of the brevetoxins (PbTx-3), yessotoxin, gymnodimine, spirolide and pectenotoxins PTX2, PTX11, at concentrations up to 1000 nM. In the ELISA format, the distinction between analogues was more apparent than on test strips. Mid-points were at 8 nM for okadaic acid, and 40 nM and 25 nM for DTX1 and DTX2, respectively.
As the causative agents of a new type of shellfish poisoning, named diarrhetic shellfish poisoning, okadaic acid, 35(S)-methylokadaic acid, 7-O-acyl derivatives of 35(S)-methylokadaic acid, two novel polyether lactones named pectenotoxin-1 and -2 have been isolated and had their structures determined. Three pectenotoxin analogues were also present. In addition to the previously identified Dinophysis fortii, D. acuminata was newly suggested as a source of toxins.
Thirteen laboratories participated in an inter-laboratory study to evaluate the method performance characteristics of a liquid chromatography-tandem mass spectrometric method (LC-MS/MS) for marine lipophilic shellfish toxins. Method performance characteristics were evaluated for mussel (Mytilus edulis), oyster (Crassostrea gigas) and cockle (Cerastoderma edule) matrices. The specific toxin analogues tested included okadaic acid (OA), dinophysistoxins-1 and -2 (DTX1, -2), azaspiracids-1, -2 and -3 (AZA1, -2, -3), pectenotoxin-2 (PTX2), yessotoxin (YTX), and 45-OH-yessotoxin (45-OH-YTX). The instrumental technique was developed as an alternative to the still widely applied biological methods (mouse or rat bioassay). Validation was conducted according to the AOAC-harmonised protocol for the design, conduct and interpretation of method-performance studies. Eight different test materials were sent as blind duplicates to the participating laboratories. Twelve laboratories returned results that were accepted to be included in the statistical evaluation. The method precision was expressed as HORRATs. For the individual toxins (except for 45-OH-YTX) HORRATs were found to be ≤1.8 (median HORRAT=0.8) in all tested materials. The recoveries of OA-, AZA- and YTX-group toxins were within the range of 80-108% and PTX2 was within the range of 62-93%. Based on the acceptable values for precision and recovery, it was concluded that the method is suitable for official control purposes to quantitatively determine OA/DTXs, AZAs, YTXs and PTX2 in shellfish.
Between March 7 and April 12, 2008, several bay systems on the east (Gulf of Mexico) coast of Texas, USA were closed to the harvesting of oysters (Crassostrea virginica) due to the presence of the DSP (Diarrheic Shellfish Poisoning) toxin okadaic acid in excess of the 20 microg/100 g tissue FDA regulatory guidance level. This was the first shellfish harvesting closure due to the confirmed presence of DSP toxins in US history. Light microscopic cell counts were performed on water samples collected from numerous sampling sites along the Texas Gulf coast where shellfish harvesting occurs. Ultra performance liquid chromatography, electrospray ionization, selected reaction monitoring, mass spectrometry (UPLC/ESI/SRM/MS) was used to detect DSP toxins in oysters. The closures were associated with an extensive bloom of the dinoflagellate Dinophysis cf. ovum. Only okadaic acid (OA) and OA acyl esters were found in shellfish tissues (max. OA eq. levels 47 microg/100 g tissue). OA was also confirmed in a bloom water sample. No illnesses were reported associated with this event. DSP toxins now add to a growing list of phycotoxins, which include those responsible for PSP (paralytic shellfish poisoning), NSP (neurotoxic shellfish poisoning), and ASP (amnesic shellfish poisoning) which must now be monitored for in US coastal waters where shellfish are harvested.
An approach involving chemical, functional and biological techniques was taken for the detection and quantification of the marine toxin okadaic acid (OA) in mussels from Thermaikos and Saronikos Gulfs, Greece, during DSP episodes that occurred in 2006-2007. Samples were analyzed using the mouse bioassay, high performance liquid chromatography with fluorimetric detection (HPLC-FLD), using l-bromoacetylpyrene (BAP), as a precolumn derivatisation reagent, and the protein phosphatase 2A inhibition assay (PP2AIA) using a commercially available kit. Okadaic acid (OA) and its polar and non-polar esters were detected and quantified by HPLC-FLD, after hydrolysis of the samples during preparation. The detection limit of the HPLC method for OA was 5.86 microg OA/kg, which permits this method to be used for the regulatory control of these toxins in shellfish. Comparison of the results by all three methods revealed excellent consistency.
Five protein serine/threonine phosphatases (PP) have been identified by cloning cDNA from mammalian and Drosophila libraries. These novel enzymes, which have not yet been detected by the techniques of protein chemistry and enzymology, are termed PPV, PP2Bw, PPX, PPY and PPZ. The complete amino acid sequences of PPX, PPY and PPZ and an almost complete sequence of PPV are presented. In the catalytic domain PPV and PPX are more similar to PP2A (57-69% identity) than PP1 (45-49% identity), while PPY and PPZ are more similar to PP1 (66-68% identity) than PP2A (44% identity). The cDNA for PP2Bw encodes a novel Ca2+/calmodulin-dependent protein phosphatase only 62% identical to PP2B in the catalytic domain. Approaches for determining the cellular functions of these protein phosphatases are discussed.
In 1961, 1971, 1976, 1979 and 1981 several cases of mussel poisoning have been recorded in the Netherlands. During the outbreak of this phenomenon, consumers of raw or cooked mussels, Mytilus edulis, obtained from the Dutch shellfish-growing areas, showed gastrointestinal disorders. Investigations revealed that phytoplankton bloom of the dinoflagellate Dinophysis acuminata Claparède & Lachman preceded the mussel poisoning. After the disappearance of these dinoflagellates, the toxicity of mussels was slowly diminishing and no longer detectable after a cleansing period of about 4 weeks at 14-15 degrees C. Toxicity of mussels could easily be detected by the rat bioassay. The chemical structure of the toxin, isolated in 1981 from toxic mussels from the Dutch Waddensea has been determined in Japan as a dinophysis-type toxin.
Ecological and health problems posed by microalgal blooms (red tides) occurring in the Southern part of Chile are reviewed. Out of the six human illnesses provoked by microalgal toxins, paralytic shellfish poisoning is the most important, because of its high mortality rate and the high levels of phycotoxins found in contaminated molluscs. Saxitoxin and its analogues bind to a receptor in the voltage-gated sodium channel of neural membranes. The most important toxin-producer microalgae are Alexandrium catenella and Dinophysis acuta. Phycotoxins become concentrated by filter-feeding shellfish, like Mytilus chilensis. Highly sensitive methods available for detection of microalgal toxins are analyzed.
The fluorescence protein phosphatase (PP-2A) inhibition assay detects okadaic acid (OA) and DTX-1 in mussels down to 1 microg/100 g of mussel tissue. It is more sensitive than the mouse bioassay (detection limit, 20 microg/100 g) or ELISA using the SCETI DSP check kit (detection limit, 10 microg/100 g). A drawback of the PP-2A assay method has been its lack of sensitivity towards the ester derivatives of OA and DTX-1. This has been addressed by including a hydrolysis step in the pretreatment of extracts which allows these derivatives to be converted to either okadaic acid or DTX-1 prior to the DSP assay. The method has been applied to the analysis of DSP in 19 samples of naturally contaminated mussels and the results from the PP-2A inhibition assay compared to those for HPLC. A good correlation was obtained for OA determined by the two methods in both unhydrolysed and hydrolysed samples. The new procedure will substantially reduce the incidence of false negatives in the DSP assay.
In 2002 several hundred people were taken ill after eating self-harvested brown crabs (Cancer pagurus) in the southern part of Norway. The symptoms were similar to diarrhetic shellfish poisoning (DSP) although with a somewhat delayed onset. This happened at the same time as an unusual early bloom of Dinophysis acuta had lead to high amounts of DSP toxins in blue mussels (Mytilus edulis) in the same area. The proposed cause of the intoxication was that crabs had accumulated toxins by eating blue mussels. Analyses of crab material from the area revealed very little free toxin in the form of okadaic acid (OA). However, after alkaline hydrolysis of the material, the amounts of OA found in the crabs were above the toxic level. MS/MS analysis of a sample from one intoxication episode indicated presence of the 14:0, 16:1, 16:0 and 18:1 fatty acid esters of okadaic acid. Esterified OA constituted more than 90% of total identified DSP toxins in crabs, indicating that not only esterified toxin from mussels was accumulated, but also that appreciable transfer of OA to OA-esters occurred in the crabs.
Marine toxins are produced by algae or bacteria and are concentrated in contaminated seafood. Substantial increases in seafood
consumption in recent years, together with globalization of the seafood trade, have increased potential exposure to these
agents. Marine toxins produce neurological, gastrointestinal, and cardiovascular syndromes, some of which result in high mortality
and long-term morbidity. Routine clinical diagnostic tests are not available for these toxins; diagnosis is based on clinical
presentation and a history of eating seafood in the preceding 24 h. There is no antidote for any of the marine toxins, and
supportive care is the mainstay of treatment. In particular, paralytic shellfish poisoning and puffer fish poisoning can cause
death within hours after consuming the toxins and may require immediate intensive care. Rapid notification of public health
authorities is essential, because timely investigation may identify the source of contaminated seafood and prevent additional
illnesses. Extensive environmental monitoring and sometimes seasonal quarantine of a harvest are employed to reduce the risk
of exposure.
When substituting the mouse bioassay for lipophilic marine algal toxins in shellfish with analytical methods, science based factors of relative toxicity for all analogues that contribute to health risk to consumers are necessary. The aim of this paper is to establish the relative intraperitoneal toxicity of dinophysistoxin-2 (DTX-2) compared with okadaic acid (OA). The study was performed as an open, randomised parallel group trial with a four level response surface design within each of the two parallels. In accordance with the response surface design model, the LD50 for DTX-2 and OA was 338 and 206 microg/kg, respectively. By use of common regression analysis, the LD50 of DTX-2 and OA were estimated to 352 microg/kg and 204 microg/kg, respectively. The deviations between the LD50 estimates by the two methods was 4% for DTX-2 and less than 1% for OA. Taken together, these results indicate that the relative toxicity of DTX-2 is about 0.6, compared to OA. Results from the PP2A assay correspond very well with the results obtained by the mouse bioassay. The IC50 concentrations for DTX-2 and OA were 5.94 and 2.81 ng/mL, respectively. This indicates that OA is about twice as toxic as DTX-2. Since inhibition of PP2A is acknowledged as the main mechanism of toxicity of the OA group toxins, this supports the establishment of a relative toxicity factor of DTX-2 of 0.6 compared with OA.
Occurrence of a new type of shellfish poisoning in the Tohoku district. Nippon Suisan Gakkaishi 1978, 44, 1249−1255. (25) Expert group: Methods for the detection of lipophilic marine biotoxins
- A Robertson
- U K London
- L K Moore
- N G Adams
Robertson, A. Diarrhetic Shellfish Poisoning, Washington, USA, 2011.
Emerging Infect. Dis. 2013, 19 (8), 1314−1316.
(24) Yasumoto, T.; Oshima, Y.; Yamaguchi, M. Occurrence of a new
type of shellfish poisoning in the Tohoku district. Nippon Suisan
Gakkaishi 1978, 44, 1249−1255.
(25) Expert group: Methods for the detection of lipophilic marine
biotoxins. London, U.K., 2007 http://collections.europarchive.org/
tna/20101209122142/http://www.food.gov.uk/multimedia/pdfs/
publication/methodslipophilic
Journal of Agricultural and Food Chemistry
Article
DOI: 10.1021/acs.jafc.5b01254
J. Agric. Food Chem. 2015, 63, 8574−8583
(26) Commission Regulation (EU). 15/2011 of 10 January 2011
amending Regulation (EC) No 2074/2005 as regards recognised
testing methods for detecting marine biotoxins in live bivalve molluscs.
Off. J. Eur. Comm. 2011, L6, 3−6.
(27) Eberhart, B.-T.; Moore, L. K.; Harrington, N.; Adams, N. G.;
DTX-2) compared with okadaic acid, based on acute intraperitoneal toxicity in mice Protein phosphatase inhibition assay adapted for determination of total DSP in contaminated mussels
- P Hess
- D O Mountfort
- T Suzuki
- P P Truman
Hess, P. Relative toxicity of dinophysistoxin-2 (DTX-2) compared
with okadaic acid, based on acute intraperitoneal toxicity in mice.
Toxicon 2007, 49 (1), 1−7.
(21) Mountfort, D. O.; Suzuki, T.; Truman, P. Protein phosphatase
inhibition assay adapted for determination of total DSP in
contaminated mussels. Toxicon 2001, 39, 383−390.
(22) van den Top, H. J.; Gerssen, A.; McCarron, P.; Van Egmond, H.
- Dis
Dis. 2005, 41, 1290−1296.
(2) Yasumoto, T.; Murata, M. Marine Toxins. Chem. Rev. 1993, 93,
1897−1909.
(3) Food and Agriculture Organization of the United Nations.
Occurrence of a new type of shellfish poisoning in the Tohoku district. Nippon Suisan Gakkaishi Expert group: Methods for the detection of lipophilic marine biotoxins as regards recognised testing methods for detecting marine biotoxins in live bivalve molluscs
- A Robertson
- Y Oshima
- M Yamaguchi
- L K Moore
- N G Adams
Robertson, A. Diarrhetic Shellfish Poisoning, Washington, USA, 2011.
Emerging Infect. Dis. 2013, 19 (8), 1314−1316.
(24) Yasumoto, T.; Oshima, Y.; Yamaguchi, M. Occurrence of a new
type of shellfish poisoning in the Tohoku district. Nippon Suisan
Gakkaishi 1978, 44, 1249−1255.
(25) Expert group: Methods for the detection of lipophilic marine
biotoxins. London, U.K., 2007 http://collections.europarchive.org/
tna/20101209122142/http://www.food.gov.uk/multimedia/pdfs/
publication/methodslipophilic
(26) Commission Regulation (EU). 15/2011 of 10 January 2011
amending Regulation (EC) No 2074/2005 as regards recognised
testing methods for detecting marine biotoxins in live bivalve molluscs.
Off. J. Eur. Comm. 2011, L6, 3−6.
(27) Eberhart, B.-T.; Moore, L. K.; Harrington, N.; Adams, N. G.;
Methods for the detection of lipophilic marine biotoxins
- U K London