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Luis M. Botana - One of the best experts on this subject based on the ideXlab platform.
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High-throughput receptor-based assay for the detection of spirolides by chemiluminescence.
Toxicon, 2013Co-Authors: Laura P. Rodríguez, Natalia Vilariño, Jordi Molgó, Rómulo Aráoz, Luis M. BotanaAbstract:Abstract The spirolides are marine toxins that belong to a new class of macrocyclic imines produced by dinoflagellates. In this study a previously described solid-phase receptor-based assay for the detection of spirolides was optimized for high-throughput screening and prevalidated. This method is based on the competition between 13-desmethyl spirolide C and biotin-α-bungarotoxin immobilized on a streptavidin-coated surface, for binding to nicotinic acetylcholine receptors. In this inhibition assay the amount of nAChR bound to the well surface is quantified using a specific antibody, followed by a second anti-mouse IgG antibody labeled with horseradish peroxidase (HRP). The assay protocol was optimized for 384-well microplates, which allowed a reduction of the amount of reagents per sample and an increase of the number of samples per plate versus previously published receptor-based assays. The sensitivity of the assay for 13-desmethyl spirolide C ranged from 5 to 150 ng mL −1 . The performance of the assay in scallop extracts was adequate, with an estimated detection limit for 13-desmethyl spirolide C of 50 μg kg −1 of Shellfish Meat. The recovery rate of 13-desmethyl spirolide C for spiked samples with this assay was 80% and the inter-assay coefficient of variation was 8%. This 384-well microplate, chemiluminescence method can be used as a high-throughput screening assay to detect 13-desmethyl spirolide C in Shellfish Meat in order to reduce the number of samples to be processed through bioassays or analytical methods.
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Development of a Solid-Phase Receptor-Based Assay for the Detection of Cyclic Imines Using a Microsphere-Flow Cytometry System.
Analytical Chemistry, 2013Co-Authors: Laura Pérez Rodríguez, Natalia Vilariño, Jordi Molgó, Rómulo Aráoz, M. Carmen Louzao, Palmer Taylor, Todd T Talley, Luis M. BotanaAbstract:Biologically active macrocycles containing a cyclic imine were isolated for the first time from aquaculture sites in Nova Scotia, Canada, during the 1990s. These compounds display a "fast-acting" toxicity in the traditional mouse bioassay for lipophilic marine toxins. Our work aimed at developing receptor-based detection method for spirolides using a microsphere/flow cytometry Luminex system. For the assay two alternatives were considered as binding proteins, the Torpedo marmorata nicotinic acetylcholine receptor (nAChR) and the Lymnaea stagnalis acetylcholine binding protein (Ls-AChBP). A receptor-based inhibition assay was developed using the immobilization of nAChR or Ls-AChBP on the surface of carboxylated microspheres and the competition of cyclic imines with biotin-α-bungarotoxin (α-BTX) for binding to these proteins. The amount of biotin-α-BTX bound to the surface of the microspheres was quantified using phycoerythrin (PE)-labeled streptavidin and the fluorescence was analyzed in a Luminex 200 system. AChBP and nAChR bound to 13-desmethyl spirolide C efficiently; however the cross-reactivity profile of the nAChR for spirolides and gymnodimine more closely matched the relative toxic potencies reported for these toxins. The nAChR was selected for further assay development. A simple sample preparation protocol consisting of an extraction with acetone yielded a final extract with no matrix interference on the nAChR/microsphere-based assay for mussels, scallops and clams. This cyclic imine detection method allowed the detection of 13-desmethyl spirolide C in the range of 10-6000 μg/kg of Shellfish Meat, displaying a higher sensitivity and wider dynamic range than other receptor-based assays previously published. This microsphere-based assay provides a rapid, sensitive and easily performed screening method that could be multiplexed for the simultaneous detection of several marine toxins.
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detection of 13 19 didesmethyl c spirolide by fluorescence polarization using torpedo electrocyte membranes
Analytical Biochemistry, 2010Co-Authors: Eva S Fonfria, Natalia Vilariño, Jordi Molgó, Rómulo Aráoz, Paz Otero, Begona Espina, Carmen M Louzao, Mercedes Alvarez, Luis M. BotanaAbstract:Abstract Fluorescence polarization (FP) is a powerful tool for studying molecular interactions by monitoring changes in the apparent size of fluorescent molecules. In this paper, a previously described fluorescence polarization assay was used to detect 13,19-didesmethyl C spirolide. The assay is based on the competition of cyclic imine marine biotoxins with α-bungarotoxin for binding to nicotinic acetylcholine receptor-enriched membranes of Torpedo marmorata . The 13,19-didesmethyl C spirolide was detected in buffer and mussel matrix. The sensitivity of the assay for the 13,19-didesmethyl C spirolide and the 13-desmethyl C spirolide was similar. After an acetone/chloroform extraction of spiked mussel Meat, the average recovery rate of 13,19-didesmethyl C spirolide was 77.7 ± 1.9%. The quantification range for this toxin in mussel was 40–200 μg/kg of Shellfish Meat. This assay can be used to detect the spirolides 13,19-didesmethyl C spirolide and 13-desmethyl C spirolide, in Shellfish as a screening assay.
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Feasibility of gymnodimine and 13-desmethyl C spirolide detection by fluorescence polarization using a receptor-based assay in Shellfish matrixes.
Analytica Chimica Acta, 2010Co-Authors: Eva S Fonfria, Natalia Vilariño, Jordi Molgó, Rómulo Aráoz, Begona Espina, Mercedes Alvarez, M. Carmen Louzao, Luis M. BotanaAbstract:The detection of toxins in Shellfish through reliable methods is essential for human health preservation and prevention of economic losses in the aquaculture industry. Although no human intoxication has been unequivocally linked to gymnodimines or spirolides, these phycotoxins are highly toxic by intraperitoneal injection causing false positives in lipophilic toxin detection by the mouse bioassay. Based on the detection of molecular interactions by fluorescence polarization an inhibition assay was developed using fluorescent alpha-bungarotoxin and nicotinic acetylcholine receptor-enriched membranes of Torpedo marmorata to detect gymnodimine and 13-desmethyl C spirolide. Both toxins, classified into the cyclic imine group, inhibit the interaction of alpha-bungarotoxin with Torpedo nicotinic acetylcholine receptors in the nM range. In this study we analyze the matrix effect of four Shellfish species on the fluorescence polarization assay. Mussels, clams, cockles and scallops were extracted with acetone and sequentially partitioned with n-hexane and chloroform. The interference of these Shellfish extracts with the alpha-bungarotoxin fluorescence or its binding to the nicotinic acetylcholine receptor was lower than 11%. The average recovery rates of gymnodimine and 13-desmethyl C spirolide using these solvents were 90.6+/-7.8% and 89.6+/-3.2%, respectively with variations among species. The quantification range of this fluorescence polarization assay for gymnodimine and 13-desmethyl C spirolide in all tested species was 80-2000 microg kg(-1) and 85-700 microg kg(-1) of Shellfish Meat, respectively. This assay format can be used to detect gymnodimine and 13-desmethyl C spirolide in Shellfish as a screening assay.
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Detection of gymnodimine-A and 13-desmethyl C spirolide phycotoxins by fluorescence polarization.
Analytical Chemistry, 2009Co-Authors: Natalia Vilariño, Jordi Molgó, Rómulo Aráoz, Eva S Fonfria, Luis M. BotanaAbstract:The gymnodimines and spirolides are phycotoxins classified into a heterogeneous group of marine biocompounds called cyclic imines. Although there is no clear evidence of their toxicity to humans, gymnodimines and spirolides are highly toxic to rodents and constitute a source of false positives in lipophilic toxin detection by the mouse bioassay. Using nicotinic acetylcholine receptor-enriched membranes of Torpedo, and fluorescent alpha-bungarotoxin, we developed a fluorescence polarization assay to detect and quantify gymnodimine-A and 13-desmethyl C spirolide. The presence of these cyclic imines in solution inhibited the interaction of fluorescent-labeled alpha-bungarotoxin with nicotinic acetylcholine receptors in a concentration-dependent manner. The sensitivity of the assay is in the order of nanomolar concentrations of gymnodimine and 13-desmethyl C spirolide. Okadaic acid, yessotoxin, and brevetoxin-2, three lipophilic marine toxins, did not interfere with this assay. A suitable extraction method in Shellfish was also developed. The gymnodimine-A and 13-desmethyl C spirolide recovery rates of mussel matrix extraction with acetone/chloroform were 63.6% +/- 3.5% and 87.4% +/- 5.3%, respectively. In summary, this inhibition assay is capable of gymnodimine-A and 13-desmethyl C spirolide detection in mussel extracts with enough sensitivity and specificity to quantify these toxins in the range of 50-2000 microg/kg and 70-700 microg/kg of Shellfish Meat, respectively.
Jordi Molgó - One of the best experts on this subject based on the ideXlab platform.
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Health risk assessment related to pinnatoxins in French Shellfish
Toxicon, 2020Co-Authors: Nathalie Arnich, Jordi Molgó, Eric Abadie, Nicolas Delcourt, Valérie Fessard, Jean-marc Fremy, Vincent Hort, Emmeline Lagrange, Thomas Maignien, Marie-bénédicte PeyratAbstract:Pinnatoxins (PnTXs) are a group of emerging marine biotoxins produced by the benthic dinoflagellate Vulcanodinium rugosum, currently not regulated in Europe or in any other country in the world. In France, PnTXs were detected for the first time in 2011, in mussels from the Ingril lagoon (South of France, Mediterranean coast). Since then, analyses carried out in mussels from this lagoon have shown high concentrations of PnTXs for several months each year. PnTXs have also been detected, to a lesser extent, in mussels from other Mediterranean lagoons and on the Atlantic and Corsican coasts. In the French data, the main analog is PnTX G (low levels of PnTX A are also present in some samples). No cases of PnTXs poisoning in humans have been reported so far in France or anywhere else in the world. In mice, PnTXs induce acute neurotoxic effects, within a few minutes after oral administration. Clinical signs of toxicity include decreased mobility, paralysis of the hind legs, tremors, jumps and breathing difficulties leading to death by respiratory arrest at high doses. The French agency for food safety (ANSES) recently conducted a review of the state of knowledge related to PnTXs and V. rugosum. Based on (i) the clinical signs of toxicity in mice, (ii) the mode of action of PnTXs as nicotinic acetylcholine receptor competitive antagonists and (iii) knowledge on drugs and natural toxins with PnTX-related pharmacology, potential human symptoms have been extrapolated and proposed. In this work, a provisional acute benchmark value for PnTX G of 0.13 μg/kg bw per day has been derived from an oral acute toxicity study in mice. Based on this value and a large Shellfish Meat portion size of 400g, a concentration lower than 23 μg PnTX G/kg Shellfish Meat is not expected to result in adverse effects in humans. ANSES recommends taking into account PnTXs in the French official monitoring program for Shellfish production and identified data gaps to refine health risk assessment.
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High-throughput receptor-based assay for the detection of spirolides by chemiluminescence.
Toxicon, 2013Co-Authors: Laura P. Rodríguez, Natalia Vilariño, Jordi Molgó, Rómulo Aráoz, Luis M. BotanaAbstract:Abstract The spirolides are marine toxins that belong to a new class of macrocyclic imines produced by dinoflagellates. In this study a previously described solid-phase receptor-based assay for the detection of spirolides was optimized for high-throughput screening and prevalidated. This method is based on the competition between 13-desmethyl spirolide C and biotin-α-bungarotoxin immobilized on a streptavidin-coated surface, for binding to nicotinic acetylcholine receptors. In this inhibition assay the amount of nAChR bound to the well surface is quantified using a specific antibody, followed by a second anti-mouse IgG antibody labeled with horseradish peroxidase (HRP). The assay protocol was optimized for 384-well microplates, which allowed a reduction of the amount of reagents per sample and an increase of the number of samples per plate versus previously published receptor-based assays. The sensitivity of the assay for 13-desmethyl spirolide C ranged from 5 to 150 ng mL −1 . The performance of the assay in scallop extracts was adequate, with an estimated detection limit for 13-desmethyl spirolide C of 50 μg kg −1 of Shellfish Meat. The recovery rate of 13-desmethyl spirolide C for spiked samples with this assay was 80% and the inter-assay coefficient of variation was 8%. This 384-well microplate, chemiluminescence method can be used as a high-throughput screening assay to detect 13-desmethyl spirolide C in Shellfish Meat in order to reduce the number of samples to be processed through bioassays or analytical methods.
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Development of a Solid-Phase Receptor-Based Assay for the Detection of Cyclic Imines Using a Microsphere-Flow Cytometry System.
Analytical Chemistry, 2013Co-Authors: Laura Pérez Rodríguez, Natalia Vilariño, Jordi Molgó, Rómulo Aráoz, M. Carmen Louzao, Palmer Taylor, Todd T Talley, Luis M. BotanaAbstract:Biologically active macrocycles containing a cyclic imine were isolated for the first time from aquaculture sites in Nova Scotia, Canada, during the 1990s. These compounds display a "fast-acting" toxicity in the traditional mouse bioassay for lipophilic marine toxins. Our work aimed at developing receptor-based detection method for spirolides using a microsphere/flow cytometry Luminex system. For the assay two alternatives were considered as binding proteins, the Torpedo marmorata nicotinic acetylcholine receptor (nAChR) and the Lymnaea stagnalis acetylcholine binding protein (Ls-AChBP). A receptor-based inhibition assay was developed using the immobilization of nAChR or Ls-AChBP on the surface of carboxylated microspheres and the competition of cyclic imines with biotin-α-bungarotoxin (α-BTX) for binding to these proteins. The amount of biotin-α-BTX bound to the surface of the microspheres was quantified using phycoerythrin (PE)-labeled streptavidin and the fluorescence was analyzed in a Luminex 200 system. AChBP and nAChR bound to 13-desmethyl spirolide C efficiently; however the cross-reactivity profile of the nAChR for spirolides and gymnodimine more closely matched the relative toxic potencies reported for these toxins. The nAChR was selected for further assay development. A simple sample preparation protocol consisting of an extraction with acetone yielded a final extract with no matrix interference on the nAChR/microsphere-based assay for mussels, scallops and clams. This cyclic imine detection method allowed the detection of 13-desmethyl spirolide C in the range of 10-6000 μg/kg of Shellfish Meat, displaying a higher sensitivity and wider dynamic range than other receptor-based assays previously published. This microsphere-based assay provides a rapid, sensitive and easily performed screening method that could be multiplexed for the simultaneous detection of several marine toxins.
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detection of 13 19 didesmethyl c spirolide by fluorescence polarization using torpedo electrocyte membranes
Analytical Biochemistry, 2010Co-Authors: Eva S Fonfria, Natalia Vilariño, Jordi Molgó, Rómulo Aráoz, Paz Otero, Begona Espina, Carmen M Louzao, Mercedes Alvarez, Luis M. BotanaAbstract:Abstract Fluorescence polarization (FP) is a powerful tool for studying molecular interactions by monitoring changes in the apparent size of fluorescent molecules. In this paper, a previously described fluorescence polarization assay was used to detect 13,19-didesmethyl C spirolide. The assay is based on the competition of cyclic imine marine biotoxins with α-bungarotoxin for binding to nicotinic acetylcholine receptor-enriched membranes of Torpedo marmorata . The 13,19-didesmethyl C spirolide was detected in buffer and mussel matrix. The sensitivity of the assay for the 13,19-didesmethyl C spirolide and the 13-desmethyl C spirolide was similar. After an acetone/chloroform extraction of spiked mussel Meat, the average recovery rate of 13,19-didesmethyl C spirolide was 77.7 ± 1.9%. The quantification range for this toxin in mussel was 40–200 μg/kg of Shellfish Meat. This assay can be used to detect the spirolides 13,19-didesmethyl C spirolide and 13-desmethyl C spirolide, in Shellfish as a screening assay.
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Feasibility of gymnodimine and 13-desmethyl C spirolide detection by fluorescence polarization using a receptor-based assay in Shellfish matrixes.
Analytica Chimica Acta, 2010Co-Authors: Eva S Fonfria, Natalia Vilariño, Jordi Molgó, Rómulo Aráoz, Begona Espina, Mercedes Alvarez, M. Carmen Louzao, Luis M. BotanaAbstract:The detection of toxins in Shellfish through reliable methods is essential for human health preservation and prevention of economic losses in the aquaculture industry. Although no human intoxication has been unequivocally linked to gymnodimines or spirolides, these phycotoxins are highly toxic by intraperitoneal injection causing false positives in lipophilic toxin detection by the mouse bioassay. Based on the detection of molecular interactions by fluorescence polarization an inhibition assay was developed using fluorescent alpha-bungarotoxin and nicotinic acetylcholine receptor-enriched membranes of Torpedo marmorata to detect gymnodimine and 13-desmethyl C spirolide. Both toxins, classified into the cyclic imine group, inhibit the interaction of alpha-bungarotoxin with Torpedo nicotinic acetylcholine receptors in the nM range. In this study we analyze the matrix effect of four Shellfish species on the fluorescence polarization assay. Mussels, clams, cockles and scallops were extracted with acetone and sequentially partitioned with n-hexane and chloroform. The interference of these Shellfish extracts with the alpha-bungarotoxin fluorescence or its binding to the nicotinic acetylcholine receptor was lower than 11%. The average recovery rates of gymnodimine and 13-desmethyl C spirolide using these solvents were 90.6+/-7.8% and 89.6+/-3.2%, respectively with variations among species. The quantification range of this fluorescence polarization assay for gymnodimine and 13-desmethyl C spirolide in all tested species was 80-2000 microg kg(-1) and 85-700 microg kg(-1) of Shellfish Meat, respectively. This assay format can be used to detect gymnodimine and 13-desmethyl C spirolide in Shellfish as a screening assay.
Natalia Vilariño - One of the best experts on this subject based on the ideXlab platform.
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Toxic Action Reevaluation of Okadaic Acid, Dinophysistoxin-1 and Dinophysistoxin-2: Toxicity Equivalency Factors Based on the Oral Toxicity Study
Karger Publishers, 2018Co-Authors: Paula Abal, Natalia Vilariño, M. carmen Louzao, Toshiyuki Suzuki, Ryuichi Watanabe, Cristina Carrera, Ana m. Botana, Mercedes r. Vieytes, Luis m. BotanaAbstract:Background/Aims: Okadaic acid (OA) and the structurally related compounds dinophysistoxin-1 (DTX1) and dinophysistoxin-2 (DTX2) are marine phycotoxins that cause diarrheic Shellfish poisoning (DSP) in humans due to ingestion of contaminated Shellfish. In order to guarantee consumer protection, the regulatory authorities have defined the maximum level of DSP toxins as 160 µg OA equivalent kg-1 Shellfish Meat. For risk assessment and overall toxicity determination, knowledge of the relative toxicities of each analogue is required. In absence of enough information from human intoxications, oral toxicity in mice is the most reliable data for establishing Toxicity Equivalence Factors (TEFs). Methods: Toxins were administered to mice by gavage, after that the symptomatology and mice mortality was registered over a period of 24 h. Organ damage data were collected at necropsy and transmission electron microscopy (TEM) was used for ultrastructural studies. Toxins in urine, feces and blood were analyzed by HPLC-MS/MS. The evaluation of in vitro potencies of OA, DTX1 and DTX2 was performed by the protein phosphatase 2A (PP2A) inhibition assay. Results: Mice that received DSP toxins by gavage showed diarrhea as the main symptom. Those toxins caused similar gastrointestinal alterations as well as intestine ultrastructural changes. However, DSP toxins did not modify tight junctions to trigger diarrhea. They had different toxicokinetics and toxic potency. The lethal dose 50 (LD50) was 487 µg kg-1 bw for DTX1, 760 µg kg-1 bw for OA and 2262 µg kg-1 bw for DTX2. Therefore, the oral TEF values are: OA = 1, DTX1 = 1.5 and DTX2 = 0.3. Conclusion: This is the first comparative study of DSP toxins performed with accurate well-characterized standards and based on acute toxicity data. Results confirmed that DTX1 is more toxic than OA by oral route while DTX2 is less toxic. Hence, the current TEFs based on intraperitoneal toxicity should be modified. Also, the generally accepted toxic mode of action of this group of toxins needs to be reevaluated
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High-throughput receptor-based assay for the detection of spirolides by chemiluminescence.
Toxicon, 2013Co-Authors: Laura P. Rodríguez, Natalia Vilariño, Jordi Molgó, Rómulo Aráoz, Luis M. BotanaAbstract:Abstract The spirolides are marine toxins that belong to a new class of macrocyclic imines produced by dinoflagellates. In this study a previously described solid-phase receptor-based assay for the detection of spirolides was optimized for high-throughput screening and prevalidated. This method is based on the competition between 13-desmethyl spirolide C and biotin-α-bungarotoxin immobilized on a streptavidin-coated surface, for binding to nicotinic acetylcholine receptors. In this inhibition assay the amount of nAChR bound to the well surface is quantified using a specific antibody, followed by a second anti-mouse IgG antibody labeled with horseradish peroxidase (HRP). The assay protocol was optimized for 384-well microplates, which allowed a reduction of the amount of reagents per sample and an increase of the number of samples per plate versus previously published receptor-based assays. The sensitivity of the assay for 13-desmethyl spirolide C ranged from 5 to 150 ng mL −1 . The performance of the assay in scallop extracts was adequate, with an estimated detection limit for 13-desmethyl spirolide C of 50 μg kg −1 of Shellfish Meat. The recovery rate of 13-desmethyl spirolide C for spiked samples with this assay was 80% and the inter-assay coefficient of variation was 8%. This 384-well microplate, chemiluminescence method can be used as a high-throughput screening assay to detect 13-desmethyl spirolide C in Shellfish Meat in order to reduce the number of samples to be processed through bioassays or analytical methods.
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Development of a Solid-Phase Receptor-Based Assay for the Detection of Cyclic Imines Using a Microsphere-Flow Cytometry System.
Analytical Chemistry, 2013Co-Authors: Laura Pérez Rodríguez, Natalia Vilariño, Jordi Molgó, Rómulo Aráoz, M. Carmen Louzao, Palmer Taylor, Todd T Talley, Luis M. BotanaAbstract:Biologically active macrocycles containing a cyclic imine were isolated for the first time from aquaculture sites in Nova Scotia, Canada, during the 1990s. These compounds display a "fast-acting" toxicity in the traditional mouse bioassay for lipophilic marine toxins. Our work aimed at developing receptor-based detection method for spirolides using a microsphere/flow cytometry Luminex system. For the assay two alternatives were considered as binding proteins, the Torpedo marmorata nicotinic acetylcholine receptor (nAChR) and the Lymnaea stagnalis acetylcholine binding protein (Ls-AChBP). A receptor-based inhibition assay was developed using the immobilization of nAChR or Ls-AChBP on the surface of carboxylated microspheres and the competition of cyclic imines with biotin-α-bungarotoxin (α-BTX) for binding to these proteins. The amount of biotin-α-BTX bound to the surface of the microspheres was quantified using phycoerythrin (PE)-labeled streptavidin and the fluorescence was analyzed in a Luminex 200 system. AChBP and nAChR bound to 13-desmethyl spirolide C efficiently; however the cross-reactivity profile of the nAChR for spirolides and gymnodimine more closely matched the relative toxic potencies reported for these toxins. The nAChR was selected for further assay development. A simple sample preparation protocol consisting of an extraction with acetone yielded a final extract with no matrix interference on the nAChR/microsphere-based assay for mussels, scallops and clams. This cyclic imine detection method allowed the detection of 13-desmethyl spirolide C in the range of 10-6000 μg/kg of Shellfish Meat, displaying a higher sensitivity and wider dynamic range than other receptor-based assays previously published. This microsphere-based assay provides a rapid, sensitive and easily performed screening method that could be multiplexed for the simultaneous detection of several marine toxins.
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detection of 13 19 didesmethyl c spirolide by fluorescence polarization using torpedo electrocyte membranes
Analytical Biochemistry, 2010Co-Authors: Eva S Fonfria, Natalia Vilariño, Jordi Molgó, Rómulo Aráoz, Paz Otero, Begona Espina, Carmen M Louzao, Mercedes Alvarez, Luis M. BotanaAbstract:Abstract Fluorescence polarization (FP) is a powerful tool for studying molecular interactions by monitoring changes in the apparent size of fluorescent molecules. In this paper, a previously described fluorescence polarization assay was used to detect 13,19-didesmethyl C spirolide. The assay is based on the competition of cyclic imine marine biotoxins with α-bungarotoxin for binding to nicotinic acetylcholine receptor-enriched membranes of Torpedo marmorata . The 13,19-didesmethyl C spirolide was detected in buffer and mussel matrix. The sensitivity of the assay for the 13,19-didesmethyl C spirolide and the 13-desmethyl C spirolide was similar. After an acetone/chloroform extraction of spiked mussel Meat, the average recovery rate of 13,19-didesmethyl C spirolide was 77.7 ± 1.9%. The quantification range for this toxin in mussel was 40–200 μg/kg of Shellfish Meat. This assay can be used to detect the spirolides 13,19-didesmethyl C spirolide and 13-desmethyl C spirolide, in Shellfish as a screening assay.
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Feasibility of gymnodimine and 13-desmethyl C spirolide detection by fluorescence polarization using a receptor-based assay in Shellfish matrixes.
Analytica Chimica Acta, 2010Co-Authors: Eva S Fonfria, Natalia Vilariño, Jordi Molgó, Rómulo Aráoz, Begona Espina, Mercedes Alvarez, M. Carmen Louzao, Luis M. BotanaAbstract:The detection of toxins in Shellfish through reliable methods is essential for human health preservation and prevention of economic losses in the aquaculture industry. Although no human intoxication has been unequivocally linked to gymnodimines or spirolides, these phycotoxins are highly toxic by intraperitoneal injection causing false positives in lipophilic toxin detection by the mouse bioassay. Based on the detection of molecular interactions by fluorescence polarization an inhibition assay was developed using fluorescent alpha-bungarotoxin and nicotinic acetylcholine receptor-enriched membranes of Torpedo marmorata to detect gymnodimine and 13-desmethyl C spirolide. Both toxins, classified into the cyclic imine group, inhibit the interaction of alpha-bungarotoxin with Torpedo nicotinic acetylcholine receptors in the nM range. In this study we analyze the matrix effect of four Shellfish species on the fluorescence polarization assay. Mussels, clams, cockles and scallops were extracted with acetone and sequentially partitioned with n-hexane and chloroform. The interference of these Shellfish extracts with the alpha-bungarotoxin fluorescence or its binding to the nicotinic acetylcholine receptor was lower than 11%. The average recovery rates of gymnodimine and 13-desmethyl C spirolide using these solvents were 90.6+/-7.8% and 89.6+/-3.2%, respectively with variations among species. The quantification range of this fluorescence polarization assay for gymnodimine and 13-desmethyl C spirolide in all tested species was 80-2000 microg kg(-1) and 85-700 microg kg(-1) of Shellfish Meat, respectively. This assay format can be used to detect gymnodimine and 13-desmethyl C spirolide in Shellfish as a screening assay.
Rómulo Aráoz - One of the best experts on this subject based on the ideXlab platform.
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High-throughput receptor-based assay for the detection of spirolides by chemiluminescence.
Toxicon, 2013Co-Authors: Laura P. Rodríguez, Natalia Vilariño, Jordi Molgó, Rómulo Aráoz, Luis M. BotanaAbstract:Abstract The spirolides are marine toxins that belong to a new class of macrocyclic imines produced by dinoflagellates. In this study a previously described solid-phase receptor-based assay for the detection of spirolides was optimized for high-throughput screening and prevalidated. This method is based on the competition between 13-desmethyl spirolide C and biotin-α-bungarotoxin immobilized on a streptavidin-coated surface, for binding to nicotinic acetylcholine receptors. In this inhibition assay the amount of nAChR bound to the well surface is quantified using a specific antibody, followed by a second anti-mouse IgG antibody labeled with horseradish peroxidase (HRP). The assay protocol was optimized for 384-well microplates, which allowed a reduction of the amount of reagents per sample and an increase of the number of samples per plate versus previously published receptor-based assays. The sensitivity of the assay for 13-desmethyl spirolide C ranged from 5 to 150 ng mL −1 . The performance of the assay in scallop extracts was adequate, with an estimated detection limit for 13-desmethyl spirolide C of 50 μg kg −1 of Shellfish Meat. The recovery rate of 13-desmethyl spirolide C for spiked samples with this assay was 80% and the inter-assay coefficient of variation was 8%. This 384-well microplate, chemiluminescence method can be used as a high-throughput screening assay to detect 13-desmethyl spirolide C in Shellfish Meat in order to reduce the number of samples to be processed through bioassays or analytical methods.
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Development of a Solid-Phase Receptor-Based Assay for the Detection of Cyclic Imines Using a Microsphere-Flow Cytometry System.
Analytical Chemistry, 2013Co-Authors: Laura Pérez Rodríguez, Natalia Vilariño, Jordi Molgó, Rómulo Aráoz, M. Carmen Louzao, Palmer Taylor, Todd T Talley, Luis M. BotanaAbstract:Biologically active macrocycles containing a cyclic imine were isolated for the first time from aquaculture sites in Nova Scotia, Canada, during the 1990s. These compounds display a "fast-acting" toxicity in the traditional mouse bioassay for lipophilic marine toxins. Our work aimed at developing receptor-based detection method for spirolides using a microsphere/flow cytometry Luminex system. For the assay two alternatives were considered as binding proteins, the Torpedo marmorata nicotinic acetylcholine receptor (nAChR) and the Lymnaea stagnalis acetylcholine binding protein (Ls-AChBP). A receptor-based inhibition assay was developed using the immobilization of nAChR or Ls-AChBP on the surface of carboxylated microspheres and the competition of cyclic imines with biotin-α-bungarotoxin (α-BTX) for binding to these proteins. The amount of biotin-α-BTX bound to the surface of the microspheres was quantified using phycoerythrin (PE)-labeled streptavidin and the fluorescence was analyzed in a Luminex 200 system. AChBP and nAChR bound to 13-desmethyl spirolide C efficiently; however the cross-reactivity profile of the nAChR for spirolides and gymnodimine more closely matched the relative toxic potencies reported for these toxins. The nAChR was selected for further assay development. A simple sample preparation protocol consisting of an extraction with acetone yielded a final extract with no matrix interference on the nAChR/microsphere-based assay for mussels, scallops and clams. This cyclic imine detection method allowed the detection of 13-desmethyl spirolide C in the range of 10-6000 μg/kg of Shellfish Meat, displaying a higher sensitivity and wider dynamic range than other receptor-based assays previously published. This microsphere-based assay provides a rapid, sensitive and easily performed screening method that could be multiplexed for the simultaneous detection of several marine toxins.
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detection of 13 19 didesmethyl c spirolide by fluorescence polarization using torpedo electrocyte membranes
Analytical Biochemistry, 2010Co-Authors: Eva S Fonfria, Natalia Vilariño, Jordi Molgó, Rómulo Aráoz, Paz Otero, Begona Espina, Carmen M Louzao, Mercedes Alvarez, Luis M. BotanaAbstract:Abstract Fluorescence polarization (FP) is a powerful tool for studying molecular interactions by monitoring changes in the apparent size of fluorescent molecules. In this paper, a previously described fluorescence polarization assay was used to detect 13,19-didesmethyl C spirolide. The assay is based on the competition of cyclic imine marine biotoxins with α-bungarotoxin for binding to nicotinic acetylcholine receptor-enriched membranes of Torpedo marmorata . The 13,19-didesmethyl C spirolide was detected in buffer and mussel matrix. The sensitivity of the assay for the 13,19-didesmethyl C spirolide and the 13-desmethyl C spirolide was similar. After an acetone/chloroform extraction of spiked mussel Meat, the average recovery rate of 13,19-didesmethyl C spirolide was 77.7 ± 1.9%. The quantification range for this toxin in mussel was 40–200 μg/kg of Shellfish Meat. This assay can be used to detect the spirolides 13,19-didesmethyl C spirolide and 13-desmethyl C spirolide, in Shellfish as a screening assay.
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Feasibility of gymnodimine and 13-desmethyl C spirolide detection by fluorescence polarization using a receptor-based assay in Shellfish matrixes.
Analytica Chimica Acta, 2010Co-Authors: Eva S Fonfria, Natalia Vilariño, Jordi Molgó, Rómulo Aráoz, Begona Espina, Mercedes Alvarez, M. Carmen Louzao, Luis M. BotanaAbstract:The detection of toxins in Shellfish through reliable methods is essential for human health preservation and prevention of economic losses in the aquaculture industry. Although no human intoxication has been unequivocally linked to gymnodimines or spirolides, these phycotoxins are highly toxic by intraperitoneal injection causing false positives in lipophilic toxin detection by the mouse bioassay. Based on the detection of molecular interactions by fluorescence polarization an inhibition assay was developed using fluorescent alpha-bungarotoxin and nicotinic acetylcholine receptor-enriched membranes of Torpedo marmorata to detect gymnodimine and 13-desmethyl C spirolide. Both toxins, classified into the cyclic imine group, inhibit the interaction of alpha-bungarotoxin with Torpedo nicotinic acetylcholine receptors in the nM range. In this study we analyze the matrix effect of four Shellfish species on the fluorescence polarization assay. Mussels, clams, cockles and scallops were extracted with acetone and sequentially partitioned with n-hexane and chloroform. The interference of these Shellfish extracts with the alpha-bungarotoxin fluorescence or its binding to the nicotinic acetylcholine receptor was lower than 11%. The average recovery rates of gymnodimine and 13-desmethyl C spirolide using these solvents were 90.6+/-7.8% and 89.6+/-3.2%, respectively with variations among species. The quantification range of this fluorescence polarization assay for gymnodimine and 13-desmethyl C spirolide in all tested species was 80-2000 microg kg(-1) and 85-700 microg kg(-1) of Shellfish Meat, respectively. This assay format can be used to detect gymnodimine and 13-desmethyl C spirolide in Shellfish as a screening assay.
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Detection of gymnodimine-A and 13-desmethyl C spirolide phycotoxins by fluorescence polarization.
Analytical Chemistry, 2009Co-Authors: Natalia Vilariño, Jordi Molgó, Rómulo Aráoz, Eva S Fonfria, Luis M. BotanaAbstract:The gymnodimines and spirolides are phycotoxins classified into a heterogeneous group of marine biocompounds called cyclic imines. Although there is no clear evidence of their toxicity to humans, gymnodimines and spirolides are highly toxic to rodents and constitute a source of false positives in lipophilic toxin detection by the mouse bioassay. Using nicotinic acetylcholine receptor-enriched membranes of Torpedo, and fluorescent alpha-bungarotoxin, we developed a fluorescence polarization assay to detect and quantify gymnodimine-A and 13-desmethyl C spirolide. The presence of these cyclic imines in solution inhibited the interaction of fluorescent-labeled alpha-bungarotoxin with nicotinic acetylcholine receptors in a concentration-dependent manner. The sensitivity of the assay is in the order of nanomolar concentrations of gymnodimine and 13-desmethyl C spirolide. Okadaic acid, yessotoxin, and brevetoxin-2, three lipophilic marine toxins, did not interfere with this assay. A suitable extraction method in Shellfish was also developed. The gymnodimine-A and 13-desmethyl C spirolide recovery rates of mussel matrix extraction with acetone/chloroform were 63.6% +/- 3.5% and 87.4% +/- 5.3%, respectively. In summary, this inhibition assay is capable of gymnodimine-A and 13-desmethyl C spirolide detection in mussel extracts with enough sensitivity and specificity to quantify these toxins in the range of 50-2000 microg/kg and 70-700 microg/kg of Shellfish Meat, respectively.
Eva S Fonfria - One of the best experts on this subject based on the ideXlab platform.
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detection of 13 19 didesmethyl c spirolide by fluorescence polarization using torpedo electrocyte membranes
Analytical Biochemistry, 2010Co-Authors: Eva S Fonfria, Natalia Vilariño, Jordi Molgó, Rómulo Aráoz, Paz Otero, Begona Espina, Carmen M Louzao, Mercedes Alvarez, Luis M. BotanaAbstract:Abstract Fluorescence polarization (FP) is a powerful tool for studying molecular interactions by monitoring changes in the apparent size of fluorescent molecules. In this paper, a previously described fluorescence polarization assay was used to detect 13,19-didesmethyl C spirolide. The assay is based on the competition of cyclic imine marine biotoxins with α-bungarotoxin for binding to nicotinic acetylcholine receptor-enriched membranes of Torpedo marmorata . The 13,19-didesmethyl C spirolide was detected in buffer and mussel matrix. The sensitivity of the assay for the 13,19-didesmethyl C spirolide and the 13-desmethyl C spirolide was similar. After an acetone/chloroform extraction of spiked mussel Meat, the average recovery rate of 13,19-didesmethyl C spirolide was 77.7 ± 1.9%. The quantification range for this toxin in mussel was 40–200 μg/kg of Shellfish Meat. This assay can be used to detect the spirolides 13,19-didesmethyl C spirolide and 13-desmethyl C spirolide, in Shellfish as a screening assay.
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Feasibility of gymnodimine and 13-desmethyl C spirolide detection by fluorescence polarization using a receptor-based assay in Shellfish matrixes.
Analytica Chimica Acta, 2010Co-Authors: Eva S Fonfria, Natalia Vilariño, Jordi Molgó, Rómulo Aráoz, Begona Espina, Mercedes Alvarez, M. Carmen Louzao, Luis M. BotanaAbstract:The detection of toxins in Shellfish through reliable methods is essential for human health preservation and prevention of economic losses in the aquaculture industry. Although no human intoxication has been unequivocally linked to gymnodimines or spirolides, these phycotoxins are highly toxic by intraperitoneal injection causing false positives in lipophilic toxin detection by the mouse bioassay. Based on the detection of molecular interactions by fluorescence polarization an inhibition assay was developed using fluorescent alpha-bungarotoxin and nicotinic acetylcholine receptor-enriched membranes of Torpedo marmorata to detect gymnodimine and 13-desmethyl C spirolide. Both toxins, classified into the cyclic imine group, inhibit the interaction of alpha-bungarotoxin with Torpedo nicotinic acetylcholine receptors in the nM range. In this study we analyze the matrix effect of four Shellfish species on the fluorescence polarization assay. Mussels, clams, cockles and scallops were extracted with acetone and sequentially partitioned with n-hexane and chloroform. The interference of these Shellfish extracts with the alpha-bungarotoxin fluorescence or its binding to the nicotinic acetylcholine receptor was lower than 11%. The average recovery rates of gymnodimine and 13-desmethyl C spirolide using these solvents were 90.6+/-7.8% and 89.6+/-3.2%, respectively with variations among species. The quantification range of this fluorescence polarization assay for gymnodimine and 13-desmethyl C spirolide in all tested species was 80-2000 microg kg(-1) and 85-700 microg kg(-1) of Shellfish Meat, respectively. This assay format can be used to detect gymnodimine and 13-desmethyl C spirolide in Shellfish as a screening assay.
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Detection of gymnodimine-A and 13-desmethyl C spirolide phycotoxins by fluorescence polarization.
Analytical Chemistry, 2009Co-Authors: Natalia Vilariño, Jordi Molgó, Rómulo Aráoz, Eva S Fonfria, Luis M. BotanaAbstract:The gymnodimines and spirolides are phycotoxins classified into a heterogeneous group of marine biocompounds called cyclic imines. Although there is no clear evidence of their toxicity to humans, gymnodimines and spirolides are highly toxic to rodents and constitute a source of false positives in lipophilic toxin detection by the mouse bioassay. Using nicotinic acetylcholine receptor-enriched membranes of Torpedo, and fluorescent alpha-bungarotoxin, we developed a fluorescence polarization assay to detect and quantify gymnodimine-A and 13-desmethyl C spirolide. The presence of these cyclic imines in solution inhibited the interaction of fluorescent-labeled alpha-bungarotoxin with nicotinic acetylcholine receptors in a concentration-dependent manner. The sensitivity of the assay is in the order of nanomolar concentrations of gymnodimine and 13-desmethyl C spirolide. Okadaic acid, yessotoxin, and brevetoxin-2, three lipophilic marine toxins, did not interfere with this assay. A suitable extraction method in Shellfish was also developed. The gymnodimine-A and 13-desmethyl C spirolide recovery rates of mussel matrix extraction with acetone/chloroform were 63.6% +/- 3.5% and 87.4% +/- 5.3%, respectively. In summary, this inhibition assay is capable of gymnodimine-A and 13-desmethyl C spirolide detection in mussel extracts with enough sensitivity and specificity to quantify these toxins in the range of 50-2000 microg/kg and 70-700 microg/kg of Shellfish Meat, respectively.