Determination of paralytic shellfish toxins in shellfish by receptor binding assay: Collaborative study (original) (raw)
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Toxicon, 2002
In parallel trials with the mouse bioassay, MIST Alerte for Paralytic Shellfish Poisoning (PSP), a rapid diagnostic test for PSP, detected 100% of the toxic extracts in over 2100 regulatory samples. Toxic extracts contained at least 80 mg saxitoxin equivalents (STX equiv.) in 100 g of shellfish tissue, or more, as measured by the regulatory AOAC w mouse bioassay. Only one potentially toxic sample, which contained 78 and 86 mg STX equiv./100 g shellfish tissue in two different mouse bioassays, was recorded as negative in one replicate of MIST Alerte. All other toxic extracts among more than 2100 regulatory shellfish tissue samples were detected by MIST Alerte for PSP. The MIST Alerte for PSP also detected the majority of extracts containing PSP toxin greater than 32 mg STX equiv./100 g, which is the mouse bioassay detection limit. The MIST Alerte for PSP gave a false positive result compared to the mouse bioassay at an average rate of about 14% over all sites, although some differences were seen between sites. Further analysis by high performance liquid chromatography (HPLC) of the (false positive) extracts showed that many contained PSP toxicity in the range of 20 -40 mg STX equiv./100 g, below the level detectable by the mouse bioassay. The MIST Alerte for PSP gave false positive results from extracts containing less than 20 mg STX equiv./100 g shellfish tissue only about 6% of the time. The PSP family of toxin analogues can occur in any combination in naturally contaminated shellfish tissue and the antibody mixture in the MIST Alerte tests detect each of the different PSP toxin analogues with different efficacy. It is therefore impossible to provide an exact detection limit for the MIST Alerte that would be applicable for all possible toxin profiles. Through the experience of comparison testing with the regulatory mouse bioassay in many parts of the world, with over 2100 different samples, the MIST Alerte for PSP has proven its ability to detect all types of profiles of the PSP toxin analogues. The detection limit for MIST Alerte for PSP was about 40 mg STX equiv./100 g for the 'average' profile of PSP toxin analogues. Since the detection limit depends on the toxin profile in the individual extract, it will also vary depending on the profile of analogues most commonly found at each geographic location. This was observed in our study. Over all sites in the trials, approximately 5% of samples below 40 mg STX equiv./100 g were positive, and 5% of samples between 40 -80 mg STX equiv./100 g were negative. This is a reflection of the different analogue profiles found in naturally contaminated extracts, even after acid hydrolysis using the AOAC w extraction method. q
ELECTROPHORESIS, 2014
Fluorescence detection 46 PSP paralytic shellfish poisoning 47 PSTs Paralytic shellfish toxins 48 RBA Receptor binding assay 49 TEFs Toxicity equivalent factors 50 UV ultraviolet 51 Keywords: 52 C 4 D, CE, FLD, paralytic shellfish toxins 53 Total number of words including figure and table legends: 5,858 54 55 3 1 ABSTRACT 56 57 Paralytic shellfish toxins (PSTs) are produced by marine and freshwater microalgae and 58 accumulate in shellfish including mussels, oysters and scallops, causing possible fatalities 59 when inadvertently consumed. Monitoring of PST content of shellfish is therefore important 60 for food safety, with currently approved methods based on HPLC, using pre-or post-column 61 oxidation for fluorescence detection (HPLC-FLD). CE is an attractive alternative for screening 62 and detection of PSTs as it is compatible with miniaturization and could be implemented in 63 portable instrumentation for on-site monitoring. In this study, CE methods were developed 64 for capacitively coupled contactless conductivity (C 4 D), fluorescence detection (FLD), 65 ultraviolet (UV) absorption detection and mass spectrometry (MS) -making this first report 66 of C 4 D and FLD for PSTs detection. Because most oxidized toxins are neutral, MEKC was used 67 in combination with FLD. The developed CZE-UV and CZE-C 4 D methods provide better 68 resolution, selectivity and separation efficiency compared to CZE-MS and MEKC-FLD. The 69 sensitivity of the CZE-C 4 D and MEKC-FLD methods were superior to UV and MS, with LOD 70 values ranging from 140-715 ng/mL for CZE-C 4 D and 60.9-104 ng/mL for MEKC-FLD. With 71 the regulatory limit for shellfish samples of 800 ng/mL, the CZE-C 4 D and MEKC-FLD methods 72 were evaluated for the screening and detection of PSTs in shellfish samples. Whilst the CZE-73 C 4 D method suffered from significant interferences from the shellfish matrix, MEKC-FLD was 74 successfully used for PST screening of a periodate-oxidized mussel sample, with results 75 confirmed by HPLC-FLD. This confirms the potential of MEKC-FLD for screening of PSTs in 76 shellfish samples. 77 78 2 INTRODUCTION 79 80 4 Microalgae produce a structurally diverse array of toxins (phycotoxins) that can accumulate 81 through the food chain and affect human health. One such toxin is saxitoxin (STX) and its 82 analogues (decarbamoylsaxitoxin (dcSTX), neosaxitoxin (NEO), decarbamoylneosaxitoxin 83 (dcNEO), gonyautoxin 1 to gonyautoxin 5 (GTX1-GTX5), decarbamoylgonyautoxin 2 to 84 decarbamoylgonyautoxin 3 (dcGTX2-dcGTX3) and C toxins), collectively known as paralytic 85 shellfish toxins (PSTs) due to their neurotoxicity in mammals, including humans. At least 18 86 PSTs have been confirmed to have toxicological effects [1-4], with their toxicity relative to 87 STX indexed through the Toxicity Equivalent Factors (TEFs) [5, 6]. In freshwaters, PSTs are 88 produced by diverse range of cyanobacteria (e.g. Anabaena circinalis) [7, 8], and in marine 89 waters primarily by several species of dinoflagellates (e.g. Alexandrium tamarense, 90
Journal of Environmental Radioactivity, 2018
Paralytic shellfish poisoning (PSP) events occur regularly along the Mediterranean and Atlantic coast of Morocco, and have been responsible for several severe cases of human intoxication. Along the southern Atlantic coast of Morocco, aquaculture and intensive artisanal fishing practices have recently been particularly heavily impacted, and toxic species have been observed in increasing intensity and frequency. In the 1990's a regulatory monitoring program was established for the coastal waters off Morocco by the National Institute of Fisheries Research (INRH), to reduce the risk of intoxication with biotoxins. The regulatory monitoring is conducted weekly and includes toxic phytoplankton enumeration and identification, as well as saxitoxin (STX) analysis in seafood using the mouse bioassay (MBA). Animal testing remains the most widely used screening method for PSP toxin detection, yet its use is being reconsidered for animal-related ethical issues, as well as for practical considerations. To be able to better evaluate alternatives to animal testing, the performance of a nuclear-based radioligand-receptor binding assay (RBA) for paralytic shellfish toxins was assessed and compared with the MBA using four commercially important shellfish matrices, including cockles Cerastoderma edule, razor shells Solen marginatus, oysters Crassostrea gigas, and mussels Perna perna. Over 50 samples were collected and analysed as part of the regulatory monitoring framework including a suite of monthly samples from 2017 and all samples identified as toxic by MBA since 2011. Testing of reference material and evaluation of assay-critical parameters (e.g. slope of calibration curve, internal quality control QC and IC50) confirmed the robustness of the RBA methodology. With this RBA method, STX-like activity detected in shellfish samples ranged from 33 to 8500 μg STX equivalents per kg. RBA data were significantly correlated (P < 0.0001, Pearson r = 0.96) with the MBA-derived dataset. Importantly, the RBA method allowed for the detection and quantification of PSP toxins at levels not detectable by using the mouse bioassay. The limits of quantification of the RBA was calculated and found to be 10-fold lower than that of the MBA, respectively 35.24 ± 5.99 and 325 μg STX equivalents per kg of tissue. In addition, the RBA was easier to use and produced reliable results more rapidly than the MBA and without use of live animals. Considering the increasing risks associated with harmful algal blooms, globally and in Morocco, together with the increased development of aquaculture production and seafood consumption and the difficulties of live animal testing, these findings indicate that the RBA method is a reliable and effective alternative to the MBA method.
Food Additives & Contaminants: Part A
The receptor-binding assay (RBA) method for the detection of paralytic shellfish poisoning (PSP) toxins was evaluated for its overall performance in comparison with the mouse bioassay (MBA). An initial study to evaluate the effects of filtering shellfish extracts prior to running the RBA indicated no significant difference between filtered and unfiltered extracts on the determined saxitoxin concentrations. Next, we tested the RBA assay on 295 naturally contaminated mussel tissue samples, ranging in concentrations from 320 μg STX equiv. kg-1 to 13,000 μg STX equiv. kg-1 by MBA. An overall trend was observed with the RBA giving higher results (256 μg STX equiv. kg-1 on average) than the MBA; however at low concentrations (< 500 μg STX equiv. kg-1) the RBA results were marginally lower. A third study was conducted using spiked mussel tissue analysed by three independent laboratories, two of which performed the RBA and one the MBA. This multi-laboratory study again showed the RBA to give higher results than the MBA; however, it also revealed that STX determination was accurate by the RBA, unlike the MBA. To optimize the assay for efficient usage under regulatory practice, three suggestions have been made: the use of an initial screening plate to separate those samples that exceed the alert level; use of rapid PSP test kits in the field and in the laboratory for screening negative samples and for early detection of toxicity; and use of an alternate commercially-available porcine membrane in place of the laboratory-prepared rat membrane homogenate. The large number of samples analysed and the diversity of the tests conducted in this study further supports the RBA as an affordable rapid method for STX detection that is also free of the routine sacrifice of live animals.
Harmful Algae, 2010
A B S T R A C T Paralytic shellfish poisoning toxins (PSP-toxins) are potent neurotoxins associated with marine dinoflagellates and may accumulate in filter-feeding shellfish to cause food intoxication in human. Monitoring programs for PSP in shellfish rely heavily on the use of traditional mouse bioassay (MBA). Considerable progress has been made in developing a reliable, rapid and relatively convenient assay for mass screening of possible PSP contaminated samples. In this study, we investigated the potential application of a commercially available antibody-based assay for routine first line screening of PSPtoxins in shellfish collected in Hong Kong. Preliminary study showed that the Jellett Rapid PSP Test (JRPT) performed acceptably in detecting regulatory limit (80 mg STXeq per 100 g of shellfish tissue) of each standard toxin under shellfish matrix mediums and human urine, except GTX1,4 and NEO. The results indicated its potential applicability in field and outbreak situations. Upon applying the kits in 938 field samples for over 2 years, no false JRPT-positive result was determined from MBA-positive samples. However, JRPT was able to detect MBA false negative sample that exceeded the regulatory limit as determined by High Performance Liquid Chromatography (HPLC). On the other hand, a number of JRPT false positive results were revealed, based on HPLC data, suggesting that the effective or actual limit of detection for JRPT was less than 40 mg STXeq per 100 g of shellfish tissue. Of all MBA-JRPT-negative samples, 21% showed HPLC-positive results. Nevertheless, the PSP toxicity levels were below the limit. No false JRPT-negative result was found during the study. This indicated its potential efficacy for use as screening assay from public health perspective. HPLC analysis showed that STX, NEO, GTX2,3 and GTX5 were the most common PSP-toxins found in shellfish. The PSP-toxins profile and their relative abundance in shellfish were demonstrated to be potentially good biochemical markers for investigating and tracing the samples' origin. In addition, 11% of samples were recorded as JRPT-invalid results, in which 78% came from oysters. Treatment of these JRPT-invalid samples by a centrifugation step followed by a centrifugal filtration was very effective (95% validity with clearer indication lines after reanalysis by JRPT) in reducing invalid results through removing potential interfering substance(s) in shellfish matrix. Complementary to MBA and HPLC toxins analysis, JRPT was shown to be an appropriate tool for rapid and mass screening of potential PSP-toxins contaminated shellfish. When public health actions are considered to verify JRPT-positive, HPLC-positive and uncertain cases samples, confirmatory test should be performed by gold method, MBA. ß
Marine Drugs, 2020
With the move away from use of mouse bioassay (MBA) to test bivalve mollusc shellfish for paralytic shellfish poisoning (PSP) toxins, countries around the world are having to adopt non-animal-based alternatives that fulfil ethical and legal requirements. Various assays have been developed which have been subjected to single-laboratory and multi-laboratory validation studies, gaining acceptance as official methods of analysis and approval for use in some countries as official control testing methods. The majority of validation studies conducted to date do not, however, incorporate shellfish species sourced from Latin America. Consequently, this study sought to investigate the performance of five alternative PSP testing methods together with the MBA, comparing the PSP toxin data generated both qualitatively and quantitatively. The methods included a receptor binding assay (RBA), two liquid chromatography with fluorescence detection (LC-FLD) methods including both pre-column and post-c...
Toxicon, 2012
Commission Regulation (EC) N 2074/2005 recognises the biological method as the reference method for Paralytic Shellfish Poisoning (PSP) toxins detection in molluscs. It was amended by Commission Regulation (EC) N 1664/2006 that accepted the so-called Lawrence method as an alternative to the reference method. The goal of this study was to compare AOAC Official Methods of Analysis 959.08 (Biological method) and 2005.06 (Prechromatographic Oxidation and Liquid Chromatography with fluorescence detection) in samples with different toxin profiles. The influence of extraction solvent in the total samples toxicity was also evaluated. A total of 40 samples including mussels, clams, scallops, razor-clams, cockles, oysters and barnacles were analysed by both official methods. Samples were selected with Alexandrium and Gymnodinium toxic profiles, from different origin and including several presentations: fresh, frozen, canned and boiled. Acetic and hydrochloric acid extractions were performed in all samples and the extracts were simultaneously analysed by both methods. Most samples were naturally contaminated and two samples were spiked. Comparison of both official methods, mouse bioassay (MBA) with HCl extraction and Liquid Chromatography with fluorescence detection (HPLC-FLD) with acetic acid extraction, led to an 85% of consistent results regarding compliance with legal limit, including samples below and above it. The linear correlation coefficient was r 2 ¼ 0.69 and the paired t test (two tails, a ¼ 0.05) indicated that there were not significant differences among both sets of data. Nevertheless, toxicity differences were found in several samples. In 15 out of 18 shellfish with a Gymnodinium toxic profile, higher toxicity levels were obtained by MBA. This fact was more evident in 7 samples, partially related to the lack of standards and the impossibility of analysing dc-NEO, C1, 2 and GTX6 at the beginning of the study. However, other factors concerning the extraction and SPE clean-ups steps may also contribute. By contrast, 9 samples presented a much higher total toxicity by HPLC-FLD than by MBA. These higher results obtained by HPLC-FLD could not only be due to the use of the highest toxicity equivalency factor (TEF) for isomers oxidated into products that coelute when total toxicity of these samples were calculated. Further analyses of results obtained by HPLC-FLD and by MBA with both extracts were done separately.
Molecules
Paralytic Shellfish Toxins (PSTs) are marine biotoxins, primarily produced by dinoflagellates of the genera Gymnodinium spp., Alexandrium spp. They can accumulate in shellfish and, through the food chain, be assimilated by humans, giving rise to Paralytic Shellfish Poisoning. The maximum permitted level for PSTs in bivalves is 800 μg STX·2HCl eqv/kg (Reg. EC N° 853/2004). Until recently, the reference analytical method was the Mouse Bioassay, but Reg. EU N° 1709/2021 entered into force on 13 October 2021 and identified in the Standard EN14526:2017 or in any other internationally recognized validated method not entailing the use of live animals as official methods. Then the official control laboratories had urgently to fulfill the new requests, face out the Mouse Bioassay and implement instrumental analytical methods. The “EURLMB SOP for the analysis of PSTs by pre-column HPLC-FLD according to OMA AOAC 2005.06” also introduced a simplified semiquantitative approach to discriminate sa...
International Journal of Food Microbiology, 1997
Consumption of shellfish contaminated with algal toxins produced by marine dinoflagellates can lead to diarrhetic shellfish poisoning (DSP). UK legislation necessitates toxin detection by mouse bioassay but this method is non-specific and lacks sensitivity. As an alternative method, an HPLC technique has been optimized, with detection limits of 0.26 lug of toxin/g of shellfish hepatopancreas for both Okadaic Acid (OA) and Dinophysistoxin-1 (DTX-1). A calorimetric protein phosphatase inhibition (PPI) assay has also been optimized. This assay detects inhibition of protein phosphatase 1 (PPIy) by OA and DTX-1 with detection limits of 1.5 ng of total toxin/g of hepatopancreas. Contaminated shellfish from several European sources, the UK monitoring programmes and mussels associated with an outbreak of DSP poisoning in the UK, have been analyzed and assessed using the two alternative methods and a commercially available enzyme-linked immunosorbent assay (ELISA) kit. The results indicate that both the HPLC and PPI assays correlate well with each other and with the UK standard mouse bioassay. In contrast, and not withstanding its advantages of rapidity and ease, the ELISA kit did not accurately and consistently detect low toxin concentrations, although it may be useful as a screening tool.