Distribution,occurrence and biotoxin composition of the main shellfish toxin producing microalgae within European waters: A comparison of methods of analysis

Harmful algal blooms (HABs) are a natural global phenomena emerging in severity and extent. Incidents have many economic, ecological and human health impacts. Monitoring and providing early warning of toxic HABs are critical for protecting public health. Current monitoring programmes include measuring the number of toxic phytoplankton cells in the water and biotoxin levels in shellfish tissue. As these efforts are demanding and labour intensive, methods which improve the efficiency are essential. This study compares the utilisation of a multitoxin surface plasmon resonance (multitoxin SPR) biosensor with enzyme-linked immunosorbent assay (ELISA) and analytical methods such as high performance liquid chromatography with fluorescence detection (HPLC-FLD) and liquid chromatography–tandem mass spectrometry (LC–MS/MS) for toxic HAB monitoring efforts in Europe. Seawater samples (n = 256) from European waters, collected 2009–2011, were analysed for biotoxins: saxitoxin and analogues, okadaic acid and dinophysistoxins 1/2 (DTX1/DTX2) and domoic acid responsible for paralytic shellfish poisoning (PSP), diarrheic shellfish poisoning (DSP) and amnesic shellfish poisoning (ASP), respectively. Biotoxins were detected mainly in samples from Spain and Ireland. France and Norway appeared to have the lowest number of toxic samples. Both the multitoxin SPR biosensor and the RNA microarray were more sensitive at detecting toxic HABs than standard light microscopy phytoplankton monitoring. Correlations between each of the detection methods were performed with the overall agreement, based on statistical 2 × 2 comparison tables, between each testing platform ranging between 32% and 74% for all three toxin families illustrating that one individual testing method may not be an ideal solution. An efficient early warning monitoring system for the detection of toxic HABs could therefore be achieved by combining both the multitoxin SPR biosensor and RNA microarray.     

Remote, subsurface detection of the algal toxin domoic acid onboard the Environmental Sample Processor: Assay development and field trials

The ability to detect harmful algal bloom (HAB) species and their toxins in real- or near real-time is a critical need for researchers studying HAB/toxin dynamics, as well as for coastal resource managers charged with monitoring bloom populations in order to mitigate their wide ranging impacts. The Environmental Sample Processor (ESP), a robotic electromechanical/fluidic system, was developed for the autonomous, subsurface application of molecular diagnostic tests and has successfully detected several HAB species using DNA probe arrays during field deployments. Since toxin production and thus the potential for public health and ecosystem effects varies considerably in natural phytoplankton populations, the concurrent detection of HAB species and their toxins onboard the ESP is essential. We describe herein the development of methods for extracting the algal toxin domoic acid (DA) from Pseudo-nitzschia cells (extraction efficiency >90%) and testing of samples using a competitive ELISA onboard the ESP. The assay detection limit is in the low ng/mL range (in extract), which corresponds to low ng/L levels of DA in seawater for a 0.5 L sample volume acquired by the ESP. We also report the first in situ detection of both a HAB organism (i.e., Pseudo-nitzschia) and its toxin, domoic acid, via the sequential (within 2–3 h) conduct of species- and toxin-specific assays during ESP deployments in Monterey Bay, CA, USA. Efforts are now underway to further refine the assay and conduct additional calibration exercises with the aim of obtaining more reliable, accurate estimates of bloom toxicity and thus their potential impacts.     

Polyclonal antibodies to domoic acid, and their use in immunoassays for domoic acid in sea water and shellfish

Ovine antibodies raised against conjugates linked through the secondary amino group of domoic acid (1) were used, together with activated-ester-derived conjugates of domoic acid (DA) as the plate coater, to develop a robust indirect competitive enzyme-linked immunosorbent assay (cELISA) for DA in shellfish and seawater. The ELISA was used to analyze shellfish samples for DA, and was compatible with several extraction procedures. The ELISA had a detection limit below 0.01 ng ml(-1), a limit of quantitation (LOQ) of 0.15 ng ml(-1) and a working range of 0.15-15 ng ml(-1) DA. The LOQ is equivalent to 38 ng g(-1) DA in shellfish flesh, assuming a 250-fold dilution during extraction. This is more than 500 times lower than the maximum permitted level (20 microg g(-1) flesh). The ELISA is designed for use alongside regulatory analyses, and, following formal validation, should be available for pre-screening of regulatory shellfish flesh samples. The ELISA was also shown to be appropriate for analysis of DA in algal cultures and in samples of seawater, and thus has the potential to provide early warning of developing algal blooms.     

Generation of High-Affinity Chicken Single-Chain Fv Antibody Fragments for Measurement of the Pseudonitzschia pungens Toxin Domoic Acid

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.     

Generation of high-affinity chicken single-chain Fv antibody fragments for measurement of the Pseudonitzschia pungens toxin domoic acid

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.     

Development of a F actin-based live-cell fluorimetric microplate assay for diarrhetic shellfish toxins

A new cytotoxicity assay for detection and quantitation of diarrhetic shellfish toxins (DSP) is presented. This assay is based upon fluorimetric determination of F-actin depolymerization induced by okadaic acid (OA)-class compounds in the BE(2)-M17 neuroblastoma cell line. No interferences were observed with other marine toxins such as saxitoxin, domoic acid, or yessotoxin, thus indicating a good specificity of the assay as expected by the direct relationship between protein phosphatase inhibition and cytoskeletal changes. The proposed method is rapid (<2h) and shows a linear response in the range of 50-300 nM OA. The detection limit of the assay for crude methanolic extracts of bivalves lies between 0.2 and 1.0 microg OA per gram of digestive glands, depending on the type of samples (fresh or canned), thus being similar to that of the mouse bioassay. The performance of this assay has been evaluated by comparative analysis of 32 toxic mussel samples by the F-actin assay, mouse bioassay, HPLC and PP2A inhibition assay. Results obtained by the F-actin method showed no differences with HPLC and significant correlation with PP2A inhibition assay (r(2)=0.71). No false negative results were obtained with this new cell assay, which also showed optimum reproducibility.     

Effect of storage on amnesic shellfish poisoning (ASP) toxins in king scallops (Pecten maximus)

Since 1998, king scallops (Pecten maximus) obtained from Scottish offshore sites have been monitored for domoic acid (DA) and epi-domoic acid (epi-DA), the principal toxic compounds associated with amnesic shellfish poisoning (ASP). The presence of these toxins in king scallops harvested from Scottish waters at concentrations exceeding the current regulatory limit (20 μg g⁻¹ shellfish flesh) is a recurrent event. However, little information was available to determine the effects that different storage conditions experienced during sample transportation to the monitoring laboratory may have on the toxin concentrations, which are subsequently detected. Furthermore, the stability of DA and epi-DA in the solvents (methanol:water (1:1, v/v) and citric acid buffer (0.5 M, pH 3.2)) routinely used for their extraction from shellfish has not previously been assessed. Results from this study demonstrate that when king scallop samples were stored for 2–3 days at 12 °C, a significantly higher toxin concentration was detected in the gonad than when samples were stored at 4 °C and analysed within 48 h. This implies that monitoring programmes must consider transport and storage conditions between harvest and analysis. Stability studies showed rapid decomposition of DA and epi-DA in aqueous methanol extracts while DA and epi-DA seem acceptably stable when stored refrigerated in citrate buffer.     

Comparison of ELISA and SPR biosensor technology for the detection of paralytic shellfish poisoning toxins

An enzyme labeled immunosorbent assay (ELISA) and surface plasmon resonance (SPR) biosensor assay for the detection of paralytic shellfish poisoning (PSP) toxins were developed and a comparative evaluation was performed. A polyclonal antibody (BC67) used in both assay formats was raised to saxitoxin–jeffamine–BSA in New Zealand white rabbits. Each assay format was designed as an inhibition assay. Shellfish samples (n = 54) were evaluated by each method using two simple rapid extraction procedures and compared to the AOAC high performance liquid chromatography (HPLC) and the mouse bioassay (MBA). The results of each assay format were comparable with the HPLC and MBA methods and demonstrate that an antibody with high sensitivity and broad specificity to PSP toxins can be applied to different immunological techniques. The method of choice will depend on the end-users needs. The reduced manual labor and simplicity of operation of the SPR biosensor compared to ELISA, ease of sample extraction and superior real time semi-quantitative analysis are key features that could make this technology applicable in a high-throughput monitoring unit.     

Feeding,egg production,and egg hatching success of the copepods Acartia tonsa and Temora longicornis on diets of the toxic diatom Pseudo-nitzschia multiseries and the non-toxic diatom Pseudo-nitzschia pungens

In 1987, there was an episode of shellfish poisoning in Canada with human fatalities caused by the diatom Pseudo-nitzschia multiseries, which produced the toxin domoic acid. In order to examine whether domoic acid in this diatom serves as a grazing deterrent for copepods, we compared feeding rates, egg production rates, egg hatching success and mortality of the calanoid copepods Acartia tonsa and Temora longicornis feeding on unialgal diets of the toxic diatom P. multiseries and the similarly-sized non-toxic diatom Pseudo-nitzschia pungens. Copepods were collected in summers of 1994, 1995 and 1996 from Shediac Bay, New Brunswick, Canada, near Prince Edward Island, the site of the 1987 episode of domoic acid shellfish poisoning. Rates of ingestion of the toxic versus the non-toxic diatom by A. tonsa and T. longicornis were similar, with only one significantly different pair of values obtained in 1994, for which A. tonsa had a higher mean rate of ingestion of the toxic than the non-toxic diatom. Thus, domoic acid did not appear to retard grazing. Analyses of copepods with high performance liquid chromatography (HPLC) revealed that copepods accumulated domoic acid when feeding on P. multiseries. Egg production rates of copepods when feeding on P. multiseries and P. pungens were very low, ranging from 0 to 2.79 eggs female^–1 d^–1. There did not appear to be differential egg production or egg hatching success on diets of the toxic and non-toxic diatoms. Mortality of females on the toxic diet was low, ranging from 0 to 20%, with a mean of 13%, and there was no apparent difference between mortality of copepods feeding on toxic versus non-toxic diatoms. Egg hatching success on both diets, although based on few eggs, ranged between 22% and 76%, with a mean percentage hatching of 45%. Diets of the non-toxic diatom plus natural seawater assemblages supplemented with dissolved domoic acid, revealed similar rates and percentages when compared to previous experiments. In summary, none of the variables measured indicated adverse effects on copepods feeding on the toxic compared to the non-toxic diatom.     

Remote,subsurface detection of the algal toxin domoic acid onboard the Environmental Sample Processor: Assay development and field trials

The ability to detect harmful algal bloom (HAB) species and their toxins in real- or near real-time is a critical need for researchers studying HAB/toxin dynamics, as well as for coastal resource managers charged with monitoring bloom populations in order to mitigate their wide ranging impacts. The Environmental Sample Processor (ESP), a robotic electromechanical/fluidic system, was developed for the autonomous, subsurface application of molecular diagnostic tests and has successfully detected several HAB species using DNA probe arrays during field deployments. Since toxin production and thus the potential for public health and ecosystem effects varies considerably in natural phytoplankton populations, the concurrent detection of HAB species and their toxins onboard the ESP is essential. We describe herein the development of methods for extracting the algal toxin domoic acid (DA) from Pseudo-nitzschia cells (extraction efficiency >90%) and testing of samples using a competitive ELISA onboard the ESP. The assay detection limit is in the low ng/mL range (in extract), which corresponds to low ng/L levels of DA in seawater for a 0.5 L sample volume acquired by the ESP. We also report the first in situ detection of both a HAB organism (i.e., Pseudo-nitzschia) and its toxin, domoic acid, via the sequential (within 2–3 h) conduct of species- and toxin-specific assays during ESP deployments in Monterey Bay, CA, USA. Efforts are now underway to further refine the assay and conduct additional calibration exercises with the aim of obtaining more reliable, accurate estimates of bloom toxicity and thus their potential impacts.     

Development of a qualitative PCR method for the Alexandrium spp. (Dinophyceae) detection in contaminated mussels (Mytilus galloprovincialis)

Paralytic shellfish poisoning (PSP) is a syndrome caused by the consumption of shellfish contaminated with neurotoxins produced by organisms of the marine dinoflagellate genus Alexandrium. A. minutum is the most widespread species responsible for PSP in the Western Mediterranean basin. The standard monitoring of shellfish farms for the presence of harmful algae and related toxins usually requires the microscopic examination of phytoplankton populations, bioassays and toxin determination by HPLC. These procedures are time-consuming and require remarkable experience, thus limiting the number of specimens that can be analyzed by a single laboratory unit. Molecular biology techniques may be helpful in the detection of target microorganisms in field samples. In this study, we developed a qualitative PCR assay for the rapid detection of all potentially toxic species belonging to the Alexandrium genus and specifically A. minutum, in contaminated mussels. Alexandrium genus-specific primers were designed to target the 5.8S rDNA region, while an A. minutum species-specific primer was designed to bind in the ITS1 region. The assay was validated using several fixed seawater samples from the Mediterranean basin, which were analyzed using PCR along with standard microscopy procedures. The assay provided a rapid method for monitoring the presence of Alexandrium spp. in mussel tissues, as well as in seawater samples. The results showed that PCR is a valid, rapid alternative procedure for the detection of target phytoplankton species either in seawater or directly in mussels, where microalgae can accumulate.     

First report of a new rapid assay for diarrhetic shellfish poisoning 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.     

Recent domoic acid closures of shellfish harvest areas in Washington State inland waterways

Several species of the toxigenic diatom Pseudo-nitzschia, together with low concentrations of domoic acid (DA) in shellfish have been observed in Puget Sound, Washington State, since 1991. However, for the first time in September 2003, high-density blooms of Pseudo-nitzschia forced the closure of recreational, commercial, and tribal subsistence shellfish harvesting in Puget Sound. Here we report on the environmental conditions associated with shellfish closures in two inland waterways of Washington State during the Fall 2005. In Sequim Bay, shellfish harvest losses occurred on September 12 following the measurement of elevated macronutrient levels on September 2, and a bloom of P. pseudodelicatissima (up to 13 million cells/L) on September 9. Ambient NH₄ concentrations >12 μM (measured on September 2) were likely due to anthropogenic sources, ostensibly from sewage inputs to Sequim Bay. The closure of a Penn Cove commercial shellfish farm on October 16 was caused by a bloom of P. australis that followed a period of sustained precipitation, elevated Skagit River flow, and persistent southeasterly winds. The relative importance of a number of environmental factors, including temperature, stratification caused by rivers, and nutrient inputs, whether natural or anthropogenic, must be carefully studied in order to better understand the recent appearance of massive blooms of toxigenic Pseudo-nitzschia in the inland waterways of Washington State.     

The role of particles on the biogeochemical cycling of domoic acid and its isomers in natural water matrices

The adsorption of dissolved domoic acid (DA) and its geometrical isomers was assessed in aqueous solutions containing various types of particles. In one series of experiments carried out in coastal seawater, detectable net adsorption of 100 nM DA occurred only onto natural seawater particles (unfiltered seawater) and 0.5 g L⁻¹ chromatographic silica (18%) in 0.2 μm-filtered seawater. Some net adsorption (<5%) also occurred in the 0.5 g L⁻¹ suspension of estuarine sediment and 0.5 g L⁻¹ solution of humic acid in filtered seawater. No losses were seen in 0.5 g L⁻¹ suspensions of illite, kaolinite, montmorillonite, and silica sand. Biological degradation accounted for small losses (8–10%) in filtered seawater without particles. A second series of experiments using organic-free, <5 μm fractions of kaolinite and montmorillonite in deionized water (DIW) demonstrated that 70% of DA adsorbed onto kaolinite, but only 5% onto montmorillonite. Geometrical isomers of DA (iso-DA D, E, and F) showed negligible adsorption (0–8%) onto a variety of particles in filtered seawater, suggesting that major ions in seawater neutralize electrostatic attractions between particles and DA isomers. These results suggest that DA and its isomers are relatively hydrophilic and not particle reactive. Our data suggest that photochemical and biological degradation of dissolved DA and its isomers appears to occur in bulk surface seawater and its transport to bottom sediments must be mainly biologically driven.     

Continuous flow immunosensor for highly selective and real-time detection of sub-ppb levels of 2-hydroxybiphenyl by using surface plasmon resonance imaging

A biosensor based on surface plasmon resonance (SPR) is developed for the detection of 2-hydroxybiphenyl (HBP). A monoclonal antibody against HBP (abbreviated hereafter as HBP-mAb) is developed and used for the detection of HBP by competitive SPR-based immunoassay and enzyme linked immunosorbent assay (ELISA) methods. A novel HBP-hapten compound, HBP-bovine serum albumin conjugate (HBP-BSA), derived by binding several HBP units with BSA by an aliphatic chain spacer is used in the development of antibody and for the functionalization of immunoprobes. HBP-BSA linked to the Au surface of the SPR sensor chip undergoes inhibitive immunoreaction with HBP-mAb in the presence of free HBP. The SPR-based immunoassay provides a rapid determination (response time: approximately 20 min) of the concentration of HBP in the range of 0.1-1000 ppb (ng/ml). Regeneration of the sensor chip is gained by treating the antibody-anchored SPR sensor chip with a pepsin solution (100 ppm (microg/ml); pH 2.0) for few minutes. The SPR sensor chip is reusable for the detection of HBP for more than 20 cycles with average loss of 0.35% reactivity per regeneration step. HBP concentration is determined as low as 0.1 and 3 ppb using the SPR sensor and ELISA measurements, respectively. The developed SPR sensor for HBP is free from interference by coexisting benzo[a]pyrene (BaP), 2,4-dichlorophenoxyacetic acid (2,4-D) and benz[a]anthracene; SPR angle shift obtained to the flow of HBP is almost same irrespective to the presence or absence of a same concentration of these carcinogenic polycyclic aromatic hydrocarbons together. The SPR sensor for HBP is proved to be applicable in simultaneous detection of HBP and BaP in parallel with another SPR sensor for BaP.     

Synthesis of hydrophobic nanoparticles for real‐time lysozyme detection using surface plasmon resonance sensor

Diagnostic biomarkers such as proteins and enzymes are generally hard to detect because of the low abundance in biological fluids. To solve this problem, the advantages of surface plasmon resonance (SPR) and nanomaterial technologies have been combined. The SPR sensors are easy to prepare, no requirement of labelling and can be detected in real time. In addition, they have high specificity and sensitivity with low cost. The nanomaterials have also crucial functions such as efficiency improvement, selectivity, and sensitivity of the detection systems. In this report, an SPR‐based sensor is developed to detect lysozyme with hydrophobic poly (N‐methacryloyl‐(L)‐phenylalanine) (PMAPA) nanoparticles. The SPR sensor was first characterized by attenuated total reflection‐Fourier transform infrared, atomic force microscope, and water contact angle measurements and performed with aqueous lysozyme solutions. Various concentrations of lysozyme solution were used to calculate kinetic and affinity coefficients. The equilibrium and adsorption isotherm models of interactions between lysozyme solutions and SPR sensor were determined and the maximum reflection, association, and dissociation constants were calculated by Langmuir model as 4.87, 0.019 nM⁻¹, and 54 nM, respectively. The selectivity studies of SPR sensor were investigated with competitive agents, hemoglobin, and myoglobin. Also, the SPR sensor was used four times in adsorption/desorption/recovery cycles and results showed that, the combination of optical SPR sensor with hydrophobic ionizable PMAPA nanoparticles in one mode enabled the detection of lysozyme molecule with high accuracy, good sensivity, real‐time, label‐free, and a low‐detection limit of 0.66 nM from lysozyme solutions. Lysozyme detection in a real sample was performed by using chicken egg white to evaluate interfering molecules present in the medium.     

Evaluation of extraction methods for analysis of domoic acid in naturally contaminated shellfish from Portugal

Domoic acid (DA) is a marine neurotoxin that is somewhat unstable, particularly in acidic media. Several protocols were used to extract DA from naturally contaminated tissues of shellfish harvested in Portugal. A modified version of AOAC method no. 991.26, with a simplified 10 g extraction, was used and compared with an aqueous 50% methanol extraction. Mean recoveries were between 81 and 85% when extracts were analysed by LC on the same day of extraction. When acid extracts were frozen for 1 or 2 days recoveries lowered to 72%, and if injection was repeated on the following 3rd or 4th days only 57–65% was recovered. Relative standard deviation of recovery for these miscellaneous samples, which was between 10 and 13% on the day of extraction, increased approximately 10% each day the extract was reanalysed. On our regulatory monitoring work, we employ an aqueous 80% methanol extraction that is common with the lipid-soluble DSP toxins. We report here a mean recovery of 90±6% for this methodology. Our long-term stability studies of domoic acid in shellfish extracts showed that slow decomposition of this compound occur in filtered aqueous methanol extracts. Additionally, we also found that in frozen tissues slow decomposition is clearly observable over a time span of 1 month.     

Comparison of surface plasmon resonance and capacitive immunosensors for cancer antigen 125 detection in human serum samples

This paper presents a comparison between surface plasmon resonance (SPR) and capacitive immunosensors for a flow injection label-free detection of cancer antigen 125 (CA 125) in human serum. Anti-CA 125 was immobilized on gold surface through a self-assembled monolayer. Parameters affecting the responses of each system were optimized. Under optimal conditions, SPR provided a detection limit of 0.1 U ml⁻¹ while 0.05 U ml⁻¹ was obtained for the capacitive system. Linearity for SPR was between 0.1 and 40 U ml⁻¹ and 0.05–40 U ml⁻¹ for capacitive system. These immunosensors were applied to analyze CA 125 concentrations in human serum samples and compared with conventional enzyme linked fluorescent assay (ELFA). Both systems showed good agreement with ELFA (P < 0.05). Moreover, these immunosensors were very stable and provided good reproducible responses after regeneration, up to 32 times for SPR and 48 times for capacitive system with relative standard deviation lower than 4%. The SPR immunosensor provided advantages in term of fast response and real-time monitoring while capacitive immunosensor offered a sensitive and cost-effective method for CA 125 detection.     

GENETIC POPULATION STRUCTURE OF PSEUDO‐NITZSCHIA PUNGENS (BACILLARIOPHYCEAE) FROM THE PACIFIC NORTHWEST AND THE NORTH SEA1

Several species of the diatom Pseudo‐nitzschia produce the neurotoxin domoic acid (DA). Consumption of fish and shellfish that have accumulated this potent excitotoxin has resulted in severe illness and even death in humans, marine mammals, and seabirds. Pseudo‐nitzschia pungens (Grunow ex Cleve) Hasle is a cosmopolitan diatom commonly occurring in the waters of the Pacific Northwest (PNW) and the eastern North Atlantic, including the North Sea. However, genetic and physiological relationships among populations throughout this large geographic distribution have not been assessed. Population genetic parameters (e.g., Hardy–Weinberg equilibrium, linkage equilibrium, F_ST) calculated for P. pungens collected from the Juan de Fuca eddy region in the PNW indicated the presence of two distinct groups that were more divergent from each other than either was from a P. pungens sample from the North Sea. Geographic heterogeneity was also detected within each of the two PNW groups. These results suggested that the populations of P. pungens recently mixed in the Juan de Fuca eddy region (a seasonally retentive feature off the coasts of Washington State, USA, and Vancouver Island, Canada) but did not exchange genetic material by sexual reproduction. Alternatively, these two groups may be cryptic (morphologically identical, but reproductively isolated) species. Identifying cryptic diversity in Pseudo‐nitzschia is important for bloom prediction and aiding the identification of molecular markers that can be used for rapid detection assay development.     

A procedure for large-scale purification of domoic acid from toxic blue mussels (Mytilus edulis)

Pure domoic acid is required for use in research to investigate the biological effects of this new shellfish toxin. It may also prove to be a useful tool in studies exploring the basis of Alzheimer's disease. In this paper we describe a procedure which is effective in obtaining adequate quantities of pure domoic acid from blue mussel (Mytilus edulis). The procedure involves tissue homogenization, treatment of homogenate with chloroform and methanol, and separation of different phases with the addition of water. The aqueous-methanolic phase (upper layer) contains water soluble components including domoic acid, the chloroform phase (lower layer) contains lipoid moieties, and the interphase contains denatured proteins. The aqueous phase containing domoic acid was removed, rotory evaporated to get rid of methanol, followed by ultrafiltration to remove high molecular weight contaminants. The filtrate was lyophilized, resuspended in 1 N HCl, centrifuged and the resulting clear solution subjected to column chromatography on C18 reversed phase silica gel. Fractions containing domoic acid were pooled, and lyophilized. A brownish dry powder contained pure domoic acid with 60–65% yield from the original tissue homogenate. Another 10–15% of domoic acid was mixed with its isomer, and can be further resolved to obtain an overall recovery of 75–80% of the starting material.