Preparation of Monoclonal Antibody for Brevetoxin 1 and Development of Ic-ELISA and Colloidal Gold Strip to Detect Brevetoxin 1.

Brevetoxin-1 (BTX-1), a marine toxin mostly produced by the dinoflagellatae Karenia brevis, has caused the death of marine organisms and has had numerous toxicological effects on human health. Hence, it is very necessary to develop a rapid, economical, and reliable immunoassay method for BTX-1 detection. In this study, two kinds of complete antigen were synthesized using the succinic anhydride and isobutyl chloroformate two-step methods. Conjugate BTX-1-OVA was used as an antigen for mice immunization, and BTX-1-BSA for measuring the titer of the produced antibodies. A hybridoma cell line 6C6 stably secreting monoclonal antibody (mAb) against BTX-1 was obtained by fusing SP2/0 myeloma cells with the spleen cells from the immunized mouse. The hybridoma 6C6 was injected into the abdomen of BALB/c mice to obtain ascites, and the anti-BTX-1 mAb was harvested from ascites by precipitation with caprylic acid/ammonium sulfate (CA-AS). The anti-BTX-1 mAb was identified as an IgG1 subtype, and the cross-reactivity results showed that anti-BTX-1 mAb was highly specific to BTX-1 with the affinity of 1.06 × 108 L/mol. The indirect competitive ELISA results indicated that the linear range for BTX-1 detection was 14-263 ng/mL with IC50 of 60 ng/mL, and a detection limit of 14 ng/mL. The average recovery rate from the spiked samples was 88 ± 2% in intra-assay and 89 ± 2% in inter-assay. The limit of detection (LOD) using the colloidal gold strip was 200 ng/mL with high specificity. Therefore, the anti-BTX-1 mAb can be used to detect BTX-1 in shellfish and other related samples.

Subacute immunotoxicity of the marine phycotoxin yessotoxin in rats.

Yessotoxin (YTX) is a marine phycotoxin produced by dinoflagellates and accumulated in filter feeding shellfish. YTX content in shellfish is regulated by many food safety authorities to protect human health, although currently no human intoxication episodes have been unequivocally related to YTX presence in food. The immune system has been proposed as one of the target organs of YTX due to alterations of lymphoid tissues and cellular and humoral components. The aim of the present study was to explore subacute immunotoxicity of YTX in rats by evaluating the haematological response, inflammatory cytokine biomarkers and the presence of YTX-induced structural alterations in the spleen and thymus. The results showed that repeated administrations of YTX caused a decrease of lymphocyte percentage and an increase of neutrophil counts, a reduction in interleukine-6 (IL-6) plasmatic levels and histopathological splenic alterations in rats after four intraperitoneal injections of YTX at doses of 50 or 70 µg/kg that were administered every 4 days along a period of 15 days. Therefore, for the first time, subacute YTX-immunotoxicity is reported in rats, suggesting that repeated exposures to low amounts of YTX might also suppose a threat to human health, especially in immuno-compromised populations.

From genome-wide to candidate gene: an investigation of variation at the major histocompatibility complex in common bottlenose dolphins exposed to harmful algal blooms.

The role the major histocompatibility complex (MHC) plays in response to exposure to environmental toxins is relatively poorly understood, particularly in comparison to its well-described role in pathogen immunity. We investigated associations between MHC diversity and resistance to brevetoxins in common bottlenose dolphins (Tursiops truncatus). A previous genome-wide association study investigating an apparent difference in harmful algal bloom (HAB) resistance among dolphin populations in the Gulf of Mexico identified genetic variation associated with survival in close genomic proximity to multiple MHC class II loci. Here, we characterized genetic variation at DQA, DQB, DRA, and DRB loci in dolphins from central-west Florida and the Florida Panhandle, including dolphins that died during HABs and dolphins presumed to have survived HAB exposure. We found that DRB and DQB exhibited patterns of genetic differentiation among geographic regions that differed from neutral microsatellite loci. In addition, genetic differentiation at DRB across multiple pairwise comparisons of live and dead dolphins was greater than differentiation observed at neutral loci. Our findings at these MHC loci did not approach the strength of association with survival previously described for a nearby genetic variant. However, the results provide evidence that selective pressures at the MHC vary among dolphin populations that differ in the frequency of HAB exposure and that the overall composition of DRB variants differs between dolphin survivors and non-survivors of HABs. These results may suggest a potential role of MHC diversity in variable survival of bottlenose dolphins exposed to HABs.

Magneto-controlled electrochemical immunoassay of brevetoxin B in seafood based on guanine-functionalized graphene nanoribbons.

A facile and feasible magneto-controlled immunosensing platform was designed for sensitive electrochemical immunoassay of brevetoxin B (BTX-2) in seafood by using guanine-assembled graphene nanoribbons (GGNRs) as molecular tags on a home-made magnetic carbon paste electrode. Initially, monoclonal mouse anti-BTX-2 antibodies were covalently immobilized on the surface of magnetic beads, which were used as the immunosensing probes for the capture of BTX-2. The recognition elements were prepared by chemical modification of bovine serum albumin-BTX-2 conjugates (BTX-2-BSA) with the GGNRs. Based on a competitive-type immunoassay format, the formed magnetic immunocomplex was integrated on the electrode with an external magnet, followed by determination in pH 6.5 phosphate-buffered solution containing 2 µM Ru(bpy)(3)Cl(2). Under optimal conditions, the electrochemical signals decreased with the increasing BTX-2 concentrations in the sample. The dynamic concentration range spanned from 1.0 pg mL(-1) to 10 ng mL(-1) with a detection limit of 1.0 pg mL(-1) BTX-2. Inter- and intra-batch assay precisions were substantially improved by resorting to the GGNR manifold. The method featured unbiased identification of negative (blank) and positive samples. No significant differences at the 95% confidence level were encountered in the analysis of 12 spiked samples including S. constricta, M. senhousia and T. granosa between the electrochemical immunoassay and commercially available enzyme-linked immunosorbent assay (ELISA) for determination of BTX-2.

Gold nanoparticles-decorated amine-terminated poly(amidoamine) dendrimer for sensitive electrochemical immunoassay of brevetoxins in food samples.

A sensitive electrochemical immunosensor for the fast screening of brevetoxin B (BTX-2) in food samples was developed by means of immobilizing BTX-2-bovine serum albumin conjugate (BTX-2-BSA) on the gold nanoparticles-decorated amine-terminated poly(amidoamine) dendrimers (AuNP-PAADs). The presence of gold nanoparticles greatly improved the conductivity of the PAADs, and three-dimensional PAADs increased the surface coverage of the biomolecules on the electrode. Under optimal conditions, three types of immunosensor, i.e. with AuNPs, PAADs, or AuNP-PAADs, were used for the determination of BTX-2 in a competitive-type immunoassay format using horseradish peroxidase-labeled anti-BTX antibodies (HRP-anti-BTX-2) as trace in the H(2)O(2)-o-phenylenediamine (o-PD) system. A low detection limit (LOD) of 0.01 ng/mL and a wide dynamic working linear range of 0.03-8 ng/mL BTX-2 using AuNP-PAADs as matrices were obtained in comparison with those of only using AuNP or PAADs. Intra-batch assay precision was substantially improved by resorting to the AuNP-PAADs manifold. The proposed method features unbiased identification of negative (blank) and positive samples. No significant differences were encountered in the analysis of the spiking real samples between the electrochemical immunosensor and liquid chromatography for the determination of BTX-2. Importantly, this method provided a biocompatible immobilization and a promising immunosensing platform for analytes with small molecules in the analysis and detection of food safety.

Effects of in vitro brevetoxin exposure on apoptosis and cellular metabolism in a leukemic T cell line (Jurkat).

Harmful algal blooms (HABs) of the toxic dinoflagellate, Karenia brevis, produce red tide toxins, or brevetoxins. Significant health effects associated with red tide toxin exposure have been reported in sea life and in humans, with brevetoxins documented within immune cells from many species. The objective of this research was to investigate potential immunotoxic effects of brevetoxins using a leukemic T cell line (Jurkat) as an in vitro model system. Viability, cell proliferation, and apoptosis assays were conducted using brevetoxin congeners PbTx-2, PbTx-3, and PbTx-6. The effects of in vitro brevetoxin exposure on cell viability and cellular metabolism or proliferation were determined using trypan blue and MTT (1-(4,5-dimethylthiazol-2-yl)-3,5-diphenylformazan), respectively. Using MTT, cellular metabolic activity was decreased in Jurkat cells exposed to 5 - 10 microg/ml PbTx-2 or PbTx-6. After 3 h, no significant effects on cell viability were observed with any toxin congener in concentrations up to 10 microg/ml. Viability decreased dramatically after 24 h in cells treated with PbTx-2 or -6. Apoptosis, as measured by caspase-3 activity, was significantly increased in cells exposed to PbTx-2 or PbTx-6. In summary, brevetoxin congeners varied in effects on Jurkat cells, with PbTx-2 and PbTx-6 eliciting greater cellular effects compared to PbTx-3.

Enzyme-linked immunosorbent assay for the detection of yessotoxin and its analogues.

Polyclonal antibodies were produced for the development of competitive enzyme-linked immunoassays for use in quantifying yessotoxins in shellfish, algal cells, and culture supernatants. Immunizing and plate coating antigens were prepared by derivatization of yessotoxin either by ozonolysis or bromination and conjugation to proteins. Two assays that were the most sensitive for yessotoxin were optimized and characterized. Cross-reactivity studies indicated that the antibodies raised have broad specificity and that binding to analogues was strongly affected by changes to the A-ring and, to a lesser extent, the K-ring regions of the toxin molecule. ELISA provides a sensitive and rapid analytical method that is suitable for screening large numbers of samples and detects all the yessotoxin analogues that the European Commission currently requires shellfish to be tested for. The assay limit of quantitation for yessotoxin in whole shellfish flesh is 75 microg/kg; therefore, assay sensitivity is sufficient to measure toxin levels well below the maximum permitted level set by the European Commission. The antibodies produced can be used in additional applications such as the immunolocalization of yessotoxins in shellfish and preparation of immunoaffinity columns.

Inhalation toxicity of brevetoxin 3 in rats exposed for 5 days.

Brevetoxins are potent neurotoxins produced by the marine dinoflagellate Karenia brevis. Exposure to brevetoxins may occur during a K. brevis red tide when the compounds become aerosolized by wind and surf. This study assesses possible adverse health effects associated with short-term inhalation exposure to brevetoxin 3. Male F344/Crl/Br rats were exposed to 500 microg brevetoxin 3/m3 by nose-only inhalation for 0.5 or 2 h/d for 5 consecutive days. Control rats were sham exposed for 2 h to vehicle. Calculated deposited brevetoxin doses were 8.3 and 33 microg/kg/d for the low- and high-dose groups, respectively. At the termination of exposures, only body weights of the high-dose group (Group B) were significantly below control values. By immunohistochemistry (IHC), small numbers of splenic and peribronchiolar lymphoid tissue macrophages stained positive for brevetoxin, while nasal mucosa, liver, and brain were IHC negative for brevetoxin. No gross or microscopic lesions were observed in any tissue examined. There was no biochemical evidence of cytotoxicity or inflammation in bronchoalveolar lavage fluid. Alveolar macrophages showed some evidence of activation following brevetoxin exposure. Humoral-mediated immunity was suppressed in brevetoxin-exposed rats as indicated by a >70% reduction in splenic plaque-forming cells in brevetoxin-exposed animals compared to controls. Results suggest that the immune system may be a target of toxicity following brevetoxin inhalation. Future studies will focus on identification of a no-effect level and mechanisms underlying brevetoxin-induced immune suppression.

Effects of brevetoxins on murine myeloma SP2/O cells: aberrant cellular division.

Massive deaths of manatees (Trichechus manatus latirostris) during the red tide seasons have been attributed to brevetoxins produced by the dinoflagellate Karenia brevis (formerly Ptychodiscus breve and Gymnodinium breve). Although these toxins have been found in macrophages and lymphocytes in the lung, liver, and secondary lymphoid tissues of these animals, the molecular mechanisms of brevetoxicosis have not yet been identified. To investigate the effects of brevetoxins on immune cells, a murine myeloma cell line (SP2/O) was used as a model for in vitro studies. By adding brevetoxins to cultures of the SP2/O cells at concentrations ranging from 20 to 600 ng/ml, an apparent increase in proliferation was observed at around 2 hours post challenge as compared to the unchallenged cell cultures. This was followed by a drop in cell number at around 3 hours, suggesting an aberrant effect of brevetoxins on cellular division, the cells generated at 2 hours being apparently short-lived. In situ immunochemical staining of the SP2/O cells at 1 and 2 hour post challenge showed an accumulation of the toxins in the nucleus. A 21-kDa protein was subsequently isolated from the SP2/O cells as having brevetoxin-binding properties, and immunologically identified as p21, a nuclear factor known to down-regulate cellular proliferation through inhibition of cyclin-dependent kinases. These data are the first on a possible effect of brevetoxins on the cell cycle via binding to p21, a phenomenon that needs to be further investigated and validated in normal immune cells.

Functional assay to measure yessotoxins in contaminated mussel samples.

Yessotoxin (YTX) treatment of MCF-7 cells results in the accumulation of a 100-kDa fragment of E-cadherin (ECRA(100)) without a parallel loss of the intact protein in cytosoluble extracts. As a consequence, concentration-dependent increases in the total immunoreactivity detectable by anti-E-cadherin antibodies relative to controls (RTI) and in the relative immunoreactivity of ECRA(100) (RI) are observed. These responses have been exploited to develop a functional assay to measure YTX in samples from contaminated mussels by a three-step procedure, consisting of (i) treatment of MCF-7 cells with YTX standard in the concentration range 0-1nM and of unknown samples; (ii) preparation of cellular extracts, fractionation of proteins by polyacrylamide gel electrophoresis under denaturing conditions, and immunoblotting with anti-E-cadherin antibodies, followed by densitometric analyses of autoradiographies and calculation of RI of ECRA(100) and of RTI of the samples; and (iii) interpolation of the YTX concentrations in unknown samples on standard curves, by the RI of ECRA(100) and the RTI of the samples. The procedure has been used to measure yessotoxins in contaminated mussel samples, and the results obtained show that this functional assay is very sensitive (limit of detection of about 100ng equivalent YTX/g of digestive gland), and robust, as (i) it is insensitive to matrix effects in the range of toxin concentrations relevant for risk assessment to protect humans from exposure to YTX, (ii) calculations are based on a molecular parameter (the RI of ECRA(100)) which is not affected by errors in sample preparation, (iii) it can be performed by the use of antibodies commercially available from different companies, and (iv) it does not show an absolute need of calibration by a pure standard within each assay.