Paralytic shellfish poison (saxitoxin family) bioassays: Automated endpoint determination and standardization of the in vitro tissue culture bioassay, and comparison with the standard mouse bioassay

Jellett, J. F.; Marks, Linda; Stewart, James E.; Dorey, Maria L.; Watson-Wright, Wendy; Lawrence, James F.

doi:10.1016/0041-0101(92)90430-d

Cited by 124 publications

(68 citation statements)

References 12 publications

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“…this mechanistic approach provides a readout of cell responses which is informative in both qualitative and quantitative terms. Cell survival, in fact, depends on the presence of Stx in the materials challenging the cells, and quantitative estimates of the total content of toxin (in equivalents) can be obtained by interpolating data in a dose-response curve of the assay, using an appropriate standard toxin (Kogure et al, 1988;Gallacher and Birkbeck, 1992;Jellett et al, 1992;Manger et al, 1993).…”

Section: Cell-based Methods For the Detection Of Toxic Agentsmentioning

confidence: 99%

“…the loss of sodium conductance in excitable cells, as a consequence of their exposure to Stx, prevents membrane depolarization and the transmission of the action potential, leading to impairment of neuromuscular function in the organism (Rossini and Hess, 2010). the cell-based method developed to detect Stx capitalized the knowledge of its molecular mechanism of action in excitable cells and exploited a neuronal cell line, which then gave its name to the procedure -the neuroblastoma assay (Kogure et al, 1988;Gallacher and Birkbeck, 1992;Jellett et al, 1992;Manger et al, 1993). In this assay, cells are exposed to veratridine, leading to opening of NaV, and to ouabain, blocking Na + ,K + -AtPase and the extrusion of sodium from the cells, causing the impairment of ion homeostasis in neuroblastoma cells resulting in cell death (Fig.…”

Section: Cell-based Methods For the Detection Of Toxic Agentsmentioning

confidence: 99%

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Towards tailored assays for cell-based approaches to toxicity testing

Rossini

2012

ALTEX

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Section: Cell-based Methods For the Detection Of Toxic Agentsmentioning

confidence: 99%

Section: Cell-based Methods For the Detection Of Toxic Agentsmentioning

confidence: 99%

Towards tailored assays for cell-based approaches to toxicity testing

Rossini

2012

ALTEX

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“…The SCB activity of the negative controls at 1/8 and even 1/10 dilutions was highly variable from assay to assay, and the standard deviations were usually above 20% when several assays were considered and always overlapped the error bars for sample extract values (data not shown). Dinoflagellate extracts have shown similar lytic effects (1,12), which could also be eliminated by sample cleanup with a C 18 Sep-Pak cartridge. However, the toxicity values for these extracts remained unaltered after the cleanup, which was not the case for our bacterial extracts.…”

mentioning

confidence: 97%

Reevaluation of Production of Paralytic Shellfish Toxin by Bacteria Associated with Dinoflagellates of the Portuguese Coast

Martins

Alvito

Tavares

et al. 2003

Appl Environ Microbiol

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Paralytic shellfish toxins (PSTs) comprise a suite of potent neurotoxins that act by blocking sodium channels in nerve axons (3). These toxins may cause severe human poisoning upon consumption of contaminated shellfish. The production of PSTs by dinoflagellates has been thoroughly documented and confirmed worldwide. Strains of the dinoflagellates Alexandrium lusitanicum Balech and Gymnodinium catenatum Graham isolated from Portuguese waters have been shown by different detection methods to produce PSTs (1,6,7,16).The potential involvement of dinoflagellate-associated bacteria in PST production, specifically the autonomous synthesis of these toxins, has been addressed using bacterial strains isolated from laboratory dinoflagellate cultures (5, 10, 15) in conjunction with high-performance liquid chromatography (HPLC) (5, 15) and in vitro assay-based toxin detection (10). Two bacterial strains isolated previously by our group from A. lusitanicum and G. catenatum and identified as Pseudomonas stutzeri and Pseudomonas diminuta, respectively, were reported to produce PSTs (5, 7). Nevertheless, the autonomous production of PSTs by bacteria remains a controversial subject, and there have been several recent reports of the incorrect identification of bacterial metabolites as PSTs by HPLC analysis (2,17,18,20,21).In the context of such findings and the continuing uncertainty regarding bacterial involvement in PST toxigenesis, the present investigation was undertaken to reevaluate our previous results on the toxin content and profile in P. stutzeri and P. diminuta. Both biological in vitro (mouse neuroblastoma [MNB] assay) and chemical (HPLC) methods were employed to account for sodium channel-blocking (SCB) activity and/or compounds with the same chromatographic behavior as PSTs produced by these two bacterial isolates. Additionally, since nutritional status has been reported to influence bacterial toxicity (4), analyses for intra-and extracellular toxins under both nutrient-replete and phosphorus-limited growth conditions were conducted.For this study, P. stutzeri and P. diminuta (5, 7) were individually inoculated into 500-ml volumes of two growth media: phosphorus-limiting artificial seawater experimental (ASWE) medium (11) containing 0.5 M Na 2 HPO 4 as well as 37.5 mM sodium succinate as the sole carbon and energy source, and a complex organic seawater complete medium (SWC) (4, 11) rich in phosphate (5 g of Bacto Tryptone, 3 g of yeast extract, and 6 ml of 50% glycerol per liter of seawater). Cultures were incubated at 19 to 21°C (150 oscillations min Ϫ1 ) under 22-E m Ϫ2 s Ϫ1 irradiance until late log phase. At that time, a cell count (in CFU per milliliter) was obtained and cells were separated from growth medium by centrifugation (10,000 ϫ g, 20 min, 8°C).For toxin extraction, cell pellets were lyophilized after centrifugation and extracted with 1 ml of 0.5 N acetic acid per 100 mg of lyophilized weight. Bacterial cells were mechanically disrupted, and the suspension was centrifuged (2,000 ϫ g, 10 min, room temper...

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“…The levels of these low-molecular-weight toxins are typically measured using either bioassay or high-performance liquid chromatography methods (16,23), both of which are time-consuming, relatively low-throughput, and often expensive procedures (11,13). Immunoassays, by contrast, are more suited to highthroughput screening, while being sensitive and highly specific (30), and recent changes in European Union regulations governing the sale of shellfish (8) indicate that immunoassays are becoming more acceptable for shellfish monitoring programs.…”

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confidence: 99%

Generation of High-Affinity Chicken Single-Chain Fv Antibody Fragments for Measurement of thePseudonitzschia pungensToxin Domoic Acid

Finlay

Shaw

Reilly

et al. 2006

Appl Environ Microbiol

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Antibody-based assay systems are now accepted by regulatory authorities for detection of the toxins produced by phytoplankton that accumulate in shellfish tissues. However, the generation of suitable antibodies for sensitive assay development remains a major challenge. We have examined the potential of using the chicken immune system to generate high-affinity, high-specificity recombinant antibody fragments against phytotoxins. Following immunization of the chicken with domoic acid-bovine serum albumin, a single-chain antibody variable region (scFv) gene library was generated from single V H and V L genes isolated from the immune cells in the spleen and bone marrow. scFvs reacting with domoic acid were isolated by phage display and affinity matured by light chain shuffling, resulting in an approximate 10-fold increase in sensitivity. The isolated scFvs were effectively expressed in Escherichia coli and readily purified by affinity chromatography. They were then used to develop a convenient and sensitive indirect competitive enzyme-linked immunosorbent assay for domoic acid, with a 50% effective dose of 156 ng/ml, which could be used reliably with shellfish extracts. This study demonstrates that chickens provide a valuable model system for the simplified, rapid generation of high-affinity recombinant antibody fragments with specificity for small toxin molecules.The accurate quantification of algal toxins in environmental and food samples is critical for consumer protection. The levels of these low-molecular-weight toxins are typically measured using either bioassay or high-performance liquid chromatography methods (16, 23), both of which are time-consuming, relatively low-throughput, and often expensive procedures (11, 13). Immunoassays, by contrast, are more suited to highthroughput screening, while being sensitive and highly specific (30), and recent changes in European Union regulations governing the sale of shellfish (8) indicate that immunoassays are becoming more acceptable for shellfish monitoring programs.Immunoassay techniques have previously relied on hyperimmune polyclonal sera from rabbits, sheep, or other mammals. These are relatively easy to produce in large quantities but do not offer the consistency required for large-scale application or commercial production. More recently, monoclonal antibodies have been favored because they can be produced in unlimited supply and are more easily standardized. However, monoclonal antibodies have their own set of difficulties, as they are almost exclusively murine in origin (10) and are laborious to screen, and small mammals such as mice do not always provide the high-affinity antibody response to small hapten molecules needed for sensitive assay development (22).The limitations of traditional techniques have led several research groups to investigate the use of phage display to produce antihapten antibodies. The phage display of recombinant antibody libraries is a robust monoclonal antibody technology that is an increasingly attractive method, as it allows the ...

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Paralytic shellfish poison (saxitoxin family) bioassays: Automated endpoint determination and standardization of the in vitro tissue culture bioassay, and comparison with the standard mouse bioassay

Cited by 124 publications

References 12 publications

Towards tailored assays for cell-based approaches to toxicity testing

Towards tailored assays for cell-based approaches to toxicity testing

Reevaluation of Production of Paralytic Shellfish Toxin by Bacteria Associated with Dinoflagellates of the Portuguese Coast

Generation of High-Affinity Chicken Single-Chain Fv Antibody Fragments for Measurement of thePseudonitzschia pungensToxin Domoic Acid

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