Katrina Campbell
email: katrina.campbell@qub.ac.uk

Dorothea F K Rawn

Barbara Niedzwiadek

Chris ; Elliott

Paralytic shellfish poisoning (PSP) toxin binders for optical biosensor technology: problems and possibilities for the future, a review

Keywords: Screening -biosensor, Screening -immunoassays, Toxicology Additives/Contaminants: Phycotoxins -PSP paralytic shellfish poisoning toxins, optical biosensor, surface plasmon resonance, antibody, saxitoxin, receptor, non-antibody binders

This review examines the developments in optical biosensor technology, which uses the phenomenon of surface plasmon resonance, for the detection of PSP toxins. Optical biosensor technology measures the competitive biomolecular interaction of a specific biological recognition element or binder with a target toxin immobilized onto a sensor chip surface against toxin in a sample. Different binders such as receptors and antibodies previously employed in functional and immunological assays have been assessed. Highlighted are the difficulties in detecting this range of low molecular weight toxins, with analogues differing at four chemical substitution sites, using a single binder.

The complications that arise with the toxicity factors of each toxin relative to the parent compound, saxitoxin, for the measurement of total toxicity relative to the mouse bioassay are also considered. For antibodies, the cross-reactivity profile does not always correlate with the toxic potency but rather to the toxin structure to which it was produced. Restrictions and availability of the toxins makes alternative chemical strategies for the synthesis of protein conjugate derivatives for antibody production a difficult task. However, when two antibodies with different cross-reactivity profiles are employed, with a toxin chip surface generic to both antibodies, it was demonstrated that the cross-reactivity profile of each could be combined into a single assay format.

Difficulties with receptors for optical biosensor analysis of low molecular weight compounds are discussed as are the potential of alternative non-antibody based binders for future assay development in this area.

Introduction

Paralytic shellfish poisoning (PSP) toxins are naturally occurring potent neurotoxins synthesized by microscopic dinoflagellates, from the genus Alexandrium, Gymnodinium and Pyrodinium in marine and freshwater environments (Reis [START_REF] Costa | Receptor binding assay for paralytic shellfish poisoning toxins: optimization and interlaboratory comparison[END_REF].

Increased temperatures, sunlight, and nutrient-rich waters are considered to cause the rapid reproduction of dinoflagellate species and thereby lead to potential "harmful algal blooms". Climate change, increased ocean eutrophication and commercial shipping are believed to contribute to the increasing frequency and occurrence of these blooms worldwide [START_REF] Botana | Functional assays for marine toxins as an alternative , high-throughput screening solution to animal tests. TRAC. 28 (5): 603-611 Isolation and Structure Elucidation of New and Unusual Saxitoxin Analogues from Mussels[END_REF]. Filter feeding organisms, such as molluscan shellfish, consuming dinoflagellates may thereby accumulate the PSP toxins and potentially transfer through the trophic chain (Deeds et al., 2008). The toxins do not appear to directly harm shellfish, but are potentially lethal to humans or other consumers such as marine mammals and birds [START_REF] Huang | Direct competitive enzyme linked immunosorbent assay for saxitoxin and neosaxitoxin[END_REF].

No visible difference in appearance is observed between harmless and toxic shellfish.

As the PSP toxins are heat stable they are not destroyed by cooking but due to their solubility in water leaching into the cooking water occurs [START_REF] Michalski | Paralytic shellfish poisoning as a health risk[END_REF][START_REF] Etheridge | Paralytic shellfish poisoning: Seafood safety and human health perspectives[END_REF]) and canning procedures are reported to reduce toxin levels [START_REF] Vieites | Canning process that diminishes paralytic shellfish poison in naturally contaminated mussels (Mytilus galloprovincialis)[END_REF].

Following consumption the toxins bind with a high affinity to site 1 of the voltage dependent sodium channel α-subunit of muscle and nerve cells, blocking the influx of sodium and preventing the generation and propagation of action potentials in these excitable cells (Cestele and Catterall, 2000). Early symptoms following ingestion include tingling of the lips and tongue which then progresses to the fingers and toes followed by the loss of control of arms and legs. Difficulty in breathing ensues from paralysis of the muscles of the chest and abdomen which results in death at high toxin doses. Fatal levels of less than 1mg of the reference toxin saxitoxin (STX) have been reported [START_REF] Van Egmond | Marine Biotoxins. Food and Agriculture Organization of the United Nations Food and Nutrition paper 80[END_REF]. There is currently no antidote for intoxication with artificial respiration as the palliative care until the toxin is excreted from the body.

PSP has major implications to public health and thereby seriously threatening economic consequences in coastal communities and aquaculture industries worldwide. Therefore, globally shellfish destined for human consumption are routinely monitored for PSP toxins by regulatory bodies. The internationally accepted and European Union (EU) reference method of analysis for PSP toxins in shellfish is the AOAC accredited biological method (AOAC, 2005a) or more commonly referred to mouse bioassay. Using this method shellfish are deemed fit for consumption based on a regulatory limit which in Europe is 800µg of PSP toxins per kg of shellfish meat, a level which has been established for the prevention of acute poisoning (EC Regulation No. 853/2004). In some other countries this limit is lower (Campas et al., 2007). However, both ethical issues relating to the use of animals and technical concerns unfolding in relation to interference and sensitivity of the assay, particularly if European Food Safety Authority (EFSA) recommendations for lowering the regulatory limit are to be considered, are advancing the search for alternative methods of analysis (EFSA, 2009).

As a family group, both the chemical and biological properties of the PSP toxins categorise the task of developing an alternative method as extremely challenging from any analytical perspective. Chemically, there are greater than twenty common PSP toxins (Figure 1) that vary in structure at four main sites (R1 to R4) of the parent compound, STX (Llewellyn, 2006). The toxins are classified into groups based on the chemical substitutions at R4 as carbamate, decarbamoyl, sulfocarbamoyl and deoxydecarbamoyl. The carbamoyl and decarbamoyl toxins are the most toxic whereas the sulfocarbamoyl toxins have a much lower potency. It is interesting to note however, that in the processing of canned seafood contaminated with PSP toxins, the sulfocarbamoyl toxins may be hydrolysed to the more toxic groups which will increase the overall PSP toxicity (Christian and Luckas, 2008). In the last number of years due to advances in mass spectrometry new structural analogues are also being identified (Dell'Aversano et al., 2008). Biologically, each analogue displays a different binding affinity to the sodium channel receptors which results in different toxicities relative to saxitoxin [START_REF] Genenah | Specific toxicity of paralytic shellfish poisons[END_REF]. Therefore, the CONTAM Panel of EFSA recommended that the regulatory limit to be expressed as µg STX equivalents per kg of shellfish meat (EFSA, 2009) which was accepted by the European Community. This method of expression of total toxicity or total level of contamination is therefore a simple concept for the biological methods whereby toxin levels correlate to toxicity and therefore the regulatory limit directly. However, this causes severe complications for Toxicity equivalence factors (TEFs) (Figure 1), originally determined by [START_REF] Oshima | Post column derivatization liquid chromatography method for paralytic shellfish toxins[END_REF] and provided as supplementary material for available toxin standards by the National Reference Centre in Canada (NRC), are used for the conversion of the individual toxin amounts to STXdiHCl equivalents. However, the EFSA working group on STX have recommended new TEFs that are due to be implemented later in 2010 in Europe with the major difference being that the TEF for dcSTX will double in value.

The use of TEFs has supported the development of analytical methods for PSP toxin analysis that can then be correlated to the mouse bioassay and the regulatory limits.

Analytical methods such as high performance liquid chromatography (HPLC) with fluorescence detection, using post-column [START_REF] Oshima | Post column derivatization liquid chromatography method for paralytic shellfish toxins[END_REF][START_REF] Asp | Analysis of PSP toxins in Norwegian mussels by a post-column derivatization HPLC method[END_REF] and pre-column oxidation methods [START_REF] Lawrence | Quantitative determination of paralytic shellfish poisoning toxins in shellfish using prechromatographic oxidation and liquid chromatography with fluorescence detection[END_REF]2004;2005) [START_REF] Diener | Application of a new zwitterionic hydrophilic interaction chromatography column for the determination of paralytic shellfish poisoning toxins[END_REF]. Nevertheless, mass spectrometry methods are more suitable for confirmatory analysis rather than screening methods due to their high expenditure and the requirement of dedicated skilled scientists for processing the data for quantitative analysis. Hence, alternative screening methods are still sought that are effective and suitable for routine on site monitoring by unskilled personnel. Different functional assays, biochemical and biosensor based technologies employing various binding molecules have shown some potential as alternative screening methods but surface plasmon resonance (SPR) optical biosensor technology has shown considerable promise in this field in a relatively short period of time. This review describes SPR optical biosensor technology and the progress and problems to date in developing an SPR assay for PSP toxin detection for full acceptance as a regulatory tool. Highlighted is a feasibility study of how combining binders has the potential to overcome certain issues and discussed are the possibilities of new novel binders with this technology.

Surface plasmon resonance (SPR) optical biosensor technology

Surface Plasmon Resonance (SPR) is a powerful physical technique that measures changes in refractive index and therefore, in the resonance angle by which polarised light is reflected from a surface which is in turn related to a change in mass on that surface [START_REF] Subrahmanyam | Application of natural receptors in sensors and assays[END_REF]. It is a phenomenon that is used to measure biomolecular interactions in real-time in a label free environment. These biomolecular interactions can be used to identify the binding of two or more interactants to each other, to determine the affinity of the interactions, to measure the actual association and dissociation rates of the interaction and for the purpose of PSP toxin detection can be exploited to quantify the concentration of one of the interactants [START_REF] Karlsson | SPR for molecular interaction analysis: a review of emerging application areas[END_REF].

Optical biosensors based on SPR technology are now an established tool in biomedical research for exploring molecular interactions but are increasingly being investigated as monitoring tools by academic and industrial scientists interested in food safety for the detection of additives and contaminants.

As Biacore ® biosensors had been proven as a successful platform for detecting low level chemical contaminants and toxins in food products the Biacore Q platform was selected for assay development for the monitoring of PSP toxins (Campbell et al., 2007;Fonfria et al., 2007). This biosensor-based assay measured the real time interaction of a specific biological recognition element or binder with a target toxin immobilized onto the sensor chip surface. A typical sensorgram, a measure of the binding response against time, is illustrated in Figure 2 (Abery, 2001). The running buffer flowing over the surface established the baseline value and as the sample solution passed over the sensor surface, the profile of the molecular interaction appeared. During injection the observed binding rate was the difference between the molecules binding to the surface and molecules leaving the surface. Consequently, binding appears fastest at the beginning of the injection as binder in the sample readily interacts with the high concentration of available toxin binding partners on the sensor surface. As the injection continues the observed rate of binding decreased as fewer toxin molecules are available on the surface. After the injection was complete, the binder dissociated and was washed away by the continuous flow of buffer. The sensor surface was regenerated back to the baseline value for the analysis of the next sample (Campbell et al., 2007).

The SPR optical biosensor method for PSP toxins was designed as an inhibition assay.

A set amount of PSP binder was mixed with the sample and injected over the surface of a sensor chip to which toxin was immobilised. When no PSP toxins were present in the sample then the binder was free to bind to the immobilised toxin producing a response (Figure 3a) (Campbell et al., 2007). When PSP toxins were present in the sample then these bound to the binder which inhibited the binder from binding to the immobilised toxin on the chip surface and a lower response was produced (Figure 3b) (Campbell et al., 2007). For most biomolecules the change in response is proportional to the mass of the material that has bound to the surface. Therefore, for binders to elicit a change in response from the baseline they must be greater than 1000 daltons and hence why the low molecular weight toxins are immobilised to the surface. Using this assay format the level of binding to the surface was inversely proportional to the concentration of PSP toxins present in the sample. Figure 4 shows a typical response against concentration calibration curve. Further developments in optical SPR biosensor technology particularly the progression of multichannel and multiplex instruments such as the Biorad Proteon XPR36 (www.biorad.com) or Biacore prototype developed as part of the EU 6 th Framework Project BioCop (www.biocop.org) provide additional options for dealing with toxicity factors of toxins and cross-reactivity issues of binders. Within the EU 7 th Framework programme the project Conffidence is evaluating the BioCop multiplex optical SPR for developing a single assay for multiple toxin groups legislated by the EU and emerging toxins to EU waters (www.conffidence.eu). This multiplex prototype device is a 4 channel instrument with 4 detection spots within each channel allowing for the simultaneous analysis of up to 16 different analytes in a sample. This increases the scope of binder interactions that could be employed to detect the different PSP analogues. An ideal scenario would be to have a highly specific binder for each of the main PSP toxin groups. Each binder assay for each group could be isolated in separate channels for simultaneous detection in a sample and then not only would the analyst be able to determine if PSP toxin was present but to which group it belonged and correlate a toxic potency level for that sample.

Binders for SPR analysis of PSP toxins

A number of different types of binder have been exploited in the development of various methodologies for the detection of PSP toxins. These include receptors such as the sodium channel receptor (SCR) and saxiphilin receptor, polyclonal and monoclonal antibodies raised against various toxin analogues and chemical binders [START_REF] Van Dolah | Single-laboratory validation of the microplate receptor binding assay for paralytic shellfish toxins in shellfish[END_REF][START_REF] Llewellyn | Microtitre plate assay for paralytic shellfish toxins using saxiphilin: gauging the effects of shellfish extract matrices, salts and pH upon assay performance[END_REF][START_REF] Usleber | Immunoassay methods for paralytic shellfish poisoning toxins[END_REF]Gawley et al., 2007). The general advantages and disadvantages of binders that have been previously investigated in the development of assays for SPR analysis for PSP toxins are highlighted in Table 1.

Receptors

Receptors can be either cell membrane bound or cytoplasmic proteins that may be exploited for their ligand binding capabilities for screening methodologies based on their functional response. Sodium channel receptors (SCR) from crude rat brain membrane preparations have been used in radioligand binding assays (Wiegele and Barchi, 1978) and in recent years been configured into a microtiter plate format [START_REF] Powell | A receptor binding assay for paralytic shellfish poisoning toxins: recent advances and applications[END_REF][START_REF] Costa | Receptor binding assay for paralytic shellfish poisoning toxins: optimization and interlaboratory comparison[END_REF]. This microtitre plate format has recently undergone successful single laboratory validation [START_REF] Van Dolah | Single-laboratory validation of the microplate receptor binding assay for paralytic shellfish toxins in shellfish[END_REF]. The fact that all STX analogues bind to SCRs with affinities that vary according to their toxic potency [START_REF] Doucette | Development and preliminary validation of a microtiter plate-based receptor binding assay for paralytic shellfish poisoning toxins[END_REF] indicates that receptor-based competitive binding assays provide a measure of a sample's toxicity, irrespective of which PSP toxin(s) are present.

Saxiphilin receptor, occurring naturally in various amphibians and terrestrial invertebrates, has also been integrated into a competitive binding assay using a microtitre plate format [START_REF] Llewellyn | Microtitre plate assay for paralytic shellfish toxins using saxiphilin: gauging the effects of shellfish extract matrices, salts and pH upon assay performance[END_REF]. Llewellyn (2006) studied the behaviour of mixtures of PSP toxins in these two unrelated competitive binding assays and found that the most potent toxins affect the toxicity of the mixture to the greatest extent with the less active toxins present required to be several orders of magnitude greater for the mixture to reflect their potency. Receptor ligand binding assays using SPR technology had previously been investigated [START_REF] Gestwicki | Using receptor conformational change to detect low molecular weight analytes by surface plasmon resonance[END_REF]De Jong et al., 2005). As receptor binders for PSP toxins reflect toxicity in a similar manner to the mouse bioassay it was envisaged that they could be an ideal candidate for the development of an SPR assay to replace the mouse bioassay. Campbell and co-workers (2007) examined crude extracts of SCR from rat brains using SPR analysis but technical difficulties were encountered with the SCR in that this preparation appeared to be stable for only a short time at 4ºC which in effect was detrimental to reliability and reproducibility. Another issue with developing SPR assays for small molecular weight toxins is that when the toxin is immobilized onto the surface, the toxin may then be orientated in such a manner that it no longer locks into the binding site of the receptor pocket making the toxin unrecognizable with minimal to no binding occurring. As SPR is dependent on changes in mass, for the receptor to be immobilized on the toxin surface the toxin binding in isolation is not of a large enough mass to achieve a change in response and the signal would need to be amplified.

Amplification could be performed in the similar manner to a sandwich assay by using a secondary antibody but for small molecular weight compounds this is difficult to achieve due to limited numbers of available epitopes that are not already encapsulated in the receptor pocket. A different approach that could be investigated is to use a ligand of a high molecular weight that would complete and dissociate appropriately with the toxin for the receptor site when the receptor is immobilised on the surface [START_REF] Karlsson | Real-time competitive kinetic analysis of interactions between low molecular weight ligands in solution and surface immobilised receptors[END_REF].

This ligand would provide the molecular mass necessary for the SPR response but

Antibodies

Antibodies unlike receptors are biochemical binders and bind selectively to chemical moieties on the structure of the toxin to which they are produced. These binders do not show a similar correlation between the toxicity of a toxin and binding affinity in the same manner as receptors for PSP toxins. The difficulty in trying to detect such small molecular weight compounds using antibodies is that there are limited functional groups to conjugate a protein for antibody production based on site specificity and therefore, few chemical reactions can be utilized for immunogen synthesis. Restricted and limited availability of toxins makes the synthesis of alternative derivatives impractical unless sufficient yield can be achieved for immunogen production. The formation of the toxin protein conjugate must also reduce the toxicity of the toxin or when immunized the animal may be affected with PSP symptoms. The first antibodies produced against PSP toxins were reported in 1964 by Johnston and colleagues [START_REF] Johnston | Haptenic properties of paralytic shellfish poison conjugated to proteins by formaldehyde treatment[END_REF]. For most PSP toxin antibodies produced since then either a modification of the Mannich reaction reported in this method or reductive alkylation chemistry (Bürk et al., 1995) has been employed for the synthesis of the protein conjugate. As highlighted by Usleber and co-workers (2001) during these reactions there are three potential reactive sites for the carbamate toxins. These include the amino, guanidine and imine groups but it is difficult to characterise which is the predominant orientation of the toxin to the protein conjugate or the ratio of protein conjugation to all three sites. Immunochemical methods are frequently used for qualitative, semi-quantitative and quantitative measurements of many chemical substances with high biological impact.

These methods are well established in clinical chemistry and endocrinology and becoming in more recent years sophisticated in their use for food and environmental analysis and toxicology screening due to their ability to function effectively in complex biological matrices without major purification. From the production of the first antibodies for PSP toxins, it was decades later when immunoassays were actually utilized to monitor PSP toxins in shellfish. Immunoassay methods for PSP toxins have been reviewed in the past [START_REF] Usleber | Immunoassay methods for paralytic shellfish poisoning toxins[END_REF] and several antibody-based approaches for detecting these toxins were described then and since this time. These include radioimmunoassay (Carlson et al., 1984), immunoaffinity columns (Dietrich et al., 1998), lateral flow devices (Jellet et al., 1998;[START_REF] Laycock | Sensitivity of lateral flow tests to mixtures of saxitoxins and applications to shellfish and phytoplankton monitoring[END_REF] and numerous competitive enzyme immunoassays (EIAs). Indirect EIAs have been developed for STX (Chu and Fan, 1985;Cembella et al., 1990;[START_REF] Micheli | Production of antibodies and development of highly sensitive formats of enzyme immunoassay for saxitoxin analysis[END_REF], neosaxitoxin (NEO) (Chu et al., 1992;Bürk et al, 1995) and gonyautoxin (GTX) 2/3 (Kawatsu et al., 2001) and direct EIAs for STX and NEO [START_REF] Usleber | Effect of heterologous paralytic shellfish poisoning toxin enzyme conjugates on the cross-reactivity of a saxitoxin enzyme immunoassay[END_REF][START_REF] Schneider | Two formats of enzyme immunoassay for the detection of saxitoxin and other paralytic shellfish poisoning toxins[END_REF][START_REF] Huang | Direct competitive enzyme linked immunosorbent assay for saxitoxin and neosaxitoxin[END_REF][START_REF] Huang | Direct competitive enzyme linked immunosorbent assay for saxitoxin and neosaxitoxin[END_REF][START_REF] Dubois | Development of ELISAs for detecting domoic acid, okadaic acid, and saxitoxin and their applicability for the detection of marine toxins in samples collected in Belgium[END_REF] and GTX 2/3 (Kawatsu et al., 2001). These assays all utilise antibodies produced using the previously mentioned protein conjugation chemistries with the primary modification being the PSP toxin to which the protein is conjugated. Although several EIAs and lateral flow test kits are commercially available [START_REF] Laycock | Sensitivity of lateral flow tests to mixtures of saxitoxins and applications to shellfish and phytoplankton monitoring[END_REF]www.abraxiskits.com;www.biosense.com;www.rbiopharm.com), one shortcoming is that they are highly specific to certain toxins. This specificity, corresponding mainly to chemical structure but not proportionately to toxicity, limits an accurate measure of the total toxicity in a sample as required by regulatory authorities. For such diverse potency within this relatively large toxin group, whose small molecular weight structures are highly similar, it is extremely difficult to match the cross-reactivity profile of an antibody to the potency of each toxin for similar comparability as a receptor assay to the mouse bioassay. [START_REF] Usleber | Immunoassay methods for paralytic shellfish poisoning toxins[END_REF] states that from an immunological point of view the PSP toxin family must be sub-divided into STX (non-hydroxylated) and NEO (hydroxylated) groups. Previous ELISA studies have demonstrated that when the antibodies are specific to either group they are less specific to the other. Therefore to incorporate the full spectra of PSP toxins to an equivalent extent, antibodies produced to both the non-hydroxylated (STX) or hydroxylated toxins (NEO) will have to be utilised. Other drawbacks to these commercial kits are the high costs, primarily due to the availability and quantity of toxin required for producing either coating antigen or enzyme labels, and the potentially subjective interpretation of results.

Current progress of SPR analysis of PSP toxins using antibody binders

To date although antibodies may have specificity issues relating to the analysis of such a large toxin group they are the most successful binder in terms of sensitivity for the detection of PSP toxins. For this reason antibodies were chosen as the model binder to demonstrate the feasibility of SPR optical biosensor technology for PSP toxin analysis.

The applicability of SPR analysis to this task was demonstrated using both a monoclonal antibody (GT-13A) raised to gonyautoxin 2/3 and a polyclonal antibody (R895) raised to STX (Campbell et al., 2007). When a STX surface was employed, which can be regenerated over 1000 times for re-use conserving toxin supplies and reducing cost, these two antibodies displayed vast differences in their cross-reactivity profiles. The GT-13A binder showed a high degree of segregation between nonhydroxylated and hydroxylated toxins indicating that when samples are analysed and compared to the mouse bioassay using this binder the toxin content of a sample would be underestimated if the toxin composition is predominately the hydroxylated toxins, NEO or GTX1/4 (Campbell et al., 2007). However, PSP toxins such as C1/C2 and GTX5 displayed high specificity although their toxic potency is relatively low to STX.

Therefore, samples containing levels of these toxins will overestimate compared to the mouse bioassay. For regulatory purposes underestimation could have severe health implications to the consumer whereby contaminated material is declared safe for consumption whereas overestimation could cause detrimental economic losses to the industry through the unnecessary closure of harvesting beds. This GT-13A binder was further assessed by SPR to determine its tolerance capabilities to various shellfish matrices and PSP toxin extraction solvents (Fonfria et al., 2007). Fonfria and coworkers ( 2007) applied different extraction procedures, previously employed for PSP toxin analysis, to different shellfish species and highlighted that by either using an ethanolic extraction procedure reported by Garthwaite and co-workers (2001) or the AOAC HPLC acetic acid extraction (AOAC, 2005b) that the determination of PSP toxins in shellfish could be performed by SPR. The Garthwaite method was recommended for further evaluation based on analysis time but only 50% recovery of STX was achieved using this extraction procedure. However, samples containing C1/C2 toxins overestimated compared to HPLC and mouse bioassay results due to the low toxic potency but high specificity of this toxin. As a proof of concept this monoclonal antibody was also employed to demonstrate that SPR biosensor-based biochemical analysis could be combined with state of the art mass spectrometry chemical analysis for joint screening and confirmatory determination of PSP toxins [START_REF] Marchesini | Towards Surface Plasmon Resonance biosensing combined with bioaffinity-assisted nano HILIC Liquid Chromatography / Time-of-flight Mass Spectrometry identification of Paralytic Shellfish Poisons[END_REF].

The R895 binder showed a much more diverse but narrower pattern in cross-reactivity profile with improved sensitivities to all toxins compared to GT-13A but the hydroxlated toxins dcNEO and GTX1/4 will require further improvement. Rawn and co-workers (2009) performed an evaluation of both binders by SPR using the Garthwaite extraction procedure for analyzing 88 natural samples and compared the results to the AOAC HPLC method (AOAC, 2005b). This study demonstrated that when using this extraction procedure different shellfish matrix effects were considerable compared to a buffer curve and if a buffer curve was used for analysis would cause an overestimation in results of 50%. Therefore, natural samples were evaluated using an extracted mussel calibration curve for better correlation in recovery and matrix effects. In general the polyclonal antibody correlated better with HPLC than the monoclonal antibody due to the differences in cross-reactivity profiles. Although, it was recommended that improved antibody response to the hydroxylated toxins would be necessary if SPR was to be a replacement to the mouse bioassay for regulatory testing. As a screening tool it was suggested that this SPR method using either antibody could eliminate greater than 80% of samples from the mouse bioassay at the current regulatory limit. In relation to the three Rs of replace, reduce and refine at this stage the SPR assay may not be a Further work was then performed on the extraction procedure in order to calibrate the SPR assay using a buffer curve to eliminate the requirement of sourcing uncontaminated material and using relatively excessive quantities of toxin in the preparation of a calibration curve (Campbell et al., 2009). This was achieved in a comparative study using a second polyclonal binder raised to STX (BC67) for SPR analysis compared to EIA, HPLC and mouse bioassay (Campbell et al., 2009). This study demonstrated that the key for immunological assay development was the binder and highlighted that the advantages with using SPR over all the other methods were simplicity, ease of use and speed of analysis. This new extraction procedure using pH5 sodium acetate buffer followed by centrifugation followed by dilution and analysis was then applied to the STX antibody and single laboratory validation of this SPR method performed highly satisfactorily [START_REF] Dubois | Development of ELISAs for detecting domoic acid, okadaic acid, and saxitoxin and their applicability for the detection of marine toxins in samples collected in Belgium[END_REF]. An inter-laboratory study between seven international laboratories is currently underway. Threshold values for the assay have been discussed for regulatory purposes but this level could vary depending on geographical location and the risk management strategies that have to be considered whether by industrial or regulatory monitoring laboratories. To date there has been no reports of PSP toxins occurring in isolation but if GTX1/4 was to do so this method could then become invalid even as a screening tool due to the lack of sensitivity of this binder to this toxin. Therefore, alternative binders are still being sought with improved sensitivity towards the hydroxylated toxins and different heterologous assay A combination of two binders, one raised to STX (Campbell et al., 2007) and the other to NEO (Bürk et al., 1995), were assessed on a NEO chip surface to determine the feasibility of combining the cross-reactivity profiles of each in order to be able to detect the full range of PSP toxins in isolation at the regulatory level (Campbell et al., unpublished data). The sensitivity and specificity of each binder relative to STX and its analogues were assessed. The cross-reactivity profile of the STX and NEO antibodies on the NEO surface was favoured to the non-hydroxylated and hydroxylated toxins at the R1 position respectively (Table 2). The % cross-reactivity of STX antibody in relation to STXdiHCl was variable in relation to toxin structure with the NEO surface. Toxins with modifications in the R4 position (Figure 1) displayed the highest % cross-reactivity followed by those with modifications to the R2 and R3 position and then those toxins that are hydroxylated in the R1 position. Combinations of modifications showed an additive decrease in % cross-reactivity with the outcome for GTX1/4, which is modified at R1, R2 and R3 positions, displaying the lowest % cross-reactivity at ≤1.0%. For the NEO antibody binder the chemical reaction used to prepare the chip surfaces was different from the reaction used to prepare the toxin protein conjugate used as the immunogen. A change in orientation of the toxin conjugated to the surface could explain the relatively low antibody titres required to produce a response on this NEO chip surface in relation to the titre of this antibody reported in the ELISA study (Bürk et al., 1995). This antibody was significantly more sensitive to the R1 hydroxylated toxins Initially for the carbamate toxins different ratios of STX antibody to NEO antibody titres were assessed with the most suitable for the mix study being 1/500 and 1/50 respectively. Different % ratios of STX to NEO antibody at these dilutions were assessed using the neosaxitoxin chip surface and each of the carbamate toxins at a concentration of 10ng/ml (Figure 3). The full cross-reactivity profile was determined using an optimum 20% STX and 80% NEO antibody (Table 3) as this ratio showed the lowest differential in cross-reactivity for the carbamate toxins. On comparison with the cross-reactivity profile of STX and NEO antibody on their own with this surface the cross-reactivity profile has been amalgamated to produce a potential single assay format for all PSP toxins. With the exceptions of GTX5 and dcSTX, which will overestimate based on this cross-reactivity profile, this antibody mix correlates much better than either binder individually with the mouse bioassay toxicity factors (provided in Figure 1) for each of the toxins. This is illustrated in Table 3 as the cross-reactivity profile in STXdiHCl equivalents. Consequently, by using optical SPR biosensor analysis there is the potential to resolve cross-reactivity issues that arise using a single binder. Combining the two polyclonal antibodies STX and NEO with a common denominator of the NEO surface enabled the design of a single assay format for both hydoxylated and non-hydroxylated toxins. In addition, as the cross-reactivity is more in line with the toxicity profile for the mouse bioassay this is an advantage in having one screening test for all PSP toxins compared to two ELISA tests. This approach of mixing antibodies raised in the same species could be used for other families of chemical contaminants or toxins where there is significant variability in the cross-reactivity profile as long as there is a common denominator in the family for chip surface immobilisation.

Potential Binders for Future Optical SPR analysis of PSP toxins

A wide range of non-antibody based binders have been reviewed previously as alternative molecules for the detection of pathogens [START_REF] Ngundi | Nonantibody-based recognition: alternative molecules for detection of pathogens[END_REF]. The most promising non-antibody binders worth investigating in their role for PSP toxin analysis by SPR are chemosensors [START_REF] Bell | Supramolecular optical chemosensors for organic analytes[END_REF], molecular imprinted polymers (MIPs) [START_REF] Hall | Imprinted polymers[END_REF] and aptamers [START_REF] Tombelli | Analytical applications of aptamers[END_REF]. The advantages and disadvantages of these particular binders are highlighted in Table 4.

Chemosensors

Chemists have used a variety of approaches to design artificial receptors or chemosensors capable of selectively binding analytes. Chemosensors are molecules specifically designed for the qualitative and quantitative monitoring of analytes and are being researched in the areas of biological and analytical chemistry, medicine and environmental sciences [START_REF] Bell | Supramolecular optical chemosensors for organic analytes[END_REF][START_REF] Minkin | Chemosensors with crown ether-based receptors[END_REF]. These molecules bind to the target analyte through non-covalent interactions and produce a change in light absorption or fluorescence. Molecular structures that have displayed some potential as chemosensors are polyalcohols, crown ethers, calixarenes, helicenes, sterically geared tripods, metal complexes, pinwheels, porphyrins and fused ring heterocycles. Most chemosensors described do not display sufficient sensitivity or selectivity to their targets for practical applications, and they usually require synthetic modification for immobilisation onto a sensor surface. However, chemosensors such as crown ether sensors like calix[4]arene have been investigated as alternative binders for STX detection (Chen et al., 2007). These binders are showing considerable promise in detecting STX using fluorescence but as yet the sensitivity is five orders of magnitude lower than that achievable using the mouse bioassay (Gawley et al., 2002;Gawley et al., 2007). For naturally contaminated shellfish samples there was no correlation between the intensity of absorption and toxicity of the sample. However SPR spectroscopic detection using these binders increased the sensitivity up to 1000 fold (Chen et al., 2007). Shellfish matrixes have not as yet been evaluated.

Molecular Imprinted Polymers (MIPs)

MIPs have received controversial exposure over the past ten years from researchers and industries in their potential role for environmental and food analysis of chemical contaminants and toxins [START_REF] Lotierzo | Surface plasmon resonance sensor for domoic acid based on grafted imprinted polymer[END_REF][START_REF] Henry | Optical interrogation of molecularly imprinted polymers and development of MIP sensors: a review Anal[END_REF][START_REF] Baggiani | Molecular imprinted polymers as synthetic receptors for the analysis of myco-and phyco-toxins[END_REF] manner as either a polyclonal or monoclonal antibody. However, the mechanism of interaction or recognition of a particular analyte to the polymer is still not completely comprehended. Unlike antibodies these are fairly rigid macroscopic structures which are insoluble in any solvent. MIPs therefore are more commonly marketed and used for selectively separating molecules for clean-up and pre-concentration of analytes from complex samples but have also been demonstrated in detection assays [START_REF] Ramstrom | Food analyses using molecularly imprinted polymers[END_REF]Chianella et al., 2003). The applicability of MIPs as a surface template in the SPR analysis of domoic acid has also been demonstrated [START_REF] Lotierzo | Surface plasmon resonance sensor for domoic acid based on grafted imprinted polymer[END_REF]. Mahony and co-workers (2005) stated that combinatorial chemistry and computational molecular modelling are the most promising concepts for the development of MIPs for enhanced recognition properties. Using this technology with advances in polymeric materials, in the future there is the potential to develop and design binders with the highest selectivity to the PSP toxin family as a whole. Though the elimination of problems associated with template leaching for quantitative analysis would have to occur.

Aptamers

Aptamers, first described by [START_REF] Tuerk | Systematic evolution of ligands by exponential enrichment: RNA ligands to bacteriophage T4 DNA polymerase[END_REF], are creating a new wave of enthusiasm as alternative binders to antibodies due to progress in aptamer screening methods (Wang and Jia, 2009) and their potential in the field of aptasensors (Wilner and Zayats, 2007). Aptamers can be divided into those created from polymers of nucleic acids (RNA and DNA) or amino acids (peptides). Nucleic acid aptamers are small synthetic oligonucleotides of up to 100 base pairs that can bind specifically to a variety of targets, such as whole cells, proteins, drugs, toxins and low molecular weight compounds, with affinities in the nanomolar and sub-nanomolar ranges. They are able to bind with a high affinity and specificity to a target molecule through complementary shape interactions as a result of their three dimensional shape. Nucleic acid aptamers are produced by an in vitro process called Systematic evolution of ligands by exponential enrichment (SELEX) [START_REF] Stoltenburg | SELEX -A revolutionary method to generate high affinity nucleic acid ligands[END_REF]. These aptamers can provide high molecular discrimination in being able to distinguish differences of a methyl group between two compounds [START_REF] Jenison | High resolution molecular discrimination by RNA[END_REF]. Aptamers offer several distinct advantages compared to antibodies or receptors. They are isolated in vitro, and the targets are of a broad spectrum. Following aptamer selection, DNA aptamers with a long shelf life can be prepared with high batch consistency at a low cost by automated DNA synthesis and RNA aptamers can be prepared by simple in vitro transcription.

Aptamers are also simple to modify and introduce various reactive groups, affinity tags, or reporting moieties essential for biosensor applications. Nucleic acid aptamers have a limited number of structures compared to peptide aptamers but offer better structural stability. Protein scaffolds are an adaptation of peptide aptamers that may overcome stability issues. Peptide aptamers still require biological systems for selection purposes.

Aptamers have been demonstrated in therapeutic applications, in the field of separation chemistry, in environmental and food analysis of chemical contaminants and toxins and a number of aptasensor applications (Wilner and Zayats, 2007;Tombelli et al., 2007;[START_REF] Stead | An RNA-Aptamer-Based Assay for the Detection and Analysis of Malachite Green and Leucomalachite Green Residues in Fish Tissue[END_REF]. These binders are now developing rapidly and maybe the one binder group that in the future replaces antibodies for diagnostic and detection applications. To date, they offer the best opportunity of finding a single binder for the

Conclusion

Optical SPR biosensor analysis for PSP toxin detection has been demonstrated over the past five years as a highly effective rapid screening method which has the potential to reduce the vast number of mouse bioassays performed worldwide. This review has highlighted the key problems associated with antibody cross-reactivity in over and underestimating total toxicity and how by using a dual antibody binder system with a generic surface that there is the potential to adjust the cross-reactivity profile to help overcome this problem. Alternative non-antibody based binders however may offer a complete non-animal based methodology for the detection of PSP toxins by SPR. In addition, advances in SPR technology with the development of multiplexing multichannel instruments could help resolve some of the difficulties in binder specificity not relating to toxicity. Multiplex multichannel SPR biosensors with highly designed binders such as nucleic acid aptamers (Mok and Li, 2008) may be the way forward for not only monitoring phycotoxins but a wide variety of food additives and contaminants.

Receptors

Have a binding affinity that generally correlates with the biological response or toxicity of the toxins Can be isolated rapidly compared to antibody production

Good sensitivity

Wider scope of binding to all compounds that act on the same receptor

Have to be isolated form biological tissues and in some cases the species of origin is difficult to obtain.

May display poor stability May be temperature sensitive for analytical purposes

Monoclonal Antibodies

Constant renewable source of binder once produced with minimal batch to batch variation.

During production less immunizing agent is required compared to polyclonal production and it is possible to select for specific epitope specificities and generate antibodies against a wider range of antigens.

React with a single epitope on an antigen with high specificity.

Binding affinity for the immunizing antigen is generally better than a receptor

Less background signal than polyclonal antibodies

For production, time, effort and commitment is high and use animals On average the binding affinity of a monoclonal antibody is lower than a polyclonal antibody.

As monoclonal antibodies are highly specific to a single epitope they may lose affinity to other antigens within a group with minor molecular modifications.

The binding affinity of an antibody does not correlate with biological response for a toxin group.

Antibodies sometimes display unexpected cross-reactivity with unrelated antigens in biological matrices.

Polyclonal Antibodies

For production relatively quick and inexpensive to produce compared to monoclonal antibodies Polyclonals will recognize multiple epitopes on any one antigen and are therefore more tolerant of minor changes in the antigen than monoclonal antibodies providing better cross-reactivity to structural antigens within a group.

The recognition of multiple epitopes generally provides more robust detection.

Binding affinity for the immunizing antigen is generally better than a monoclonal antibody or receptor Animals are required and are prone to batch to batch variability.

They produce large amounts of non-specific antibodies which can sometimes give background signal in some applications.

Multiple epitopes make it important to check for any cross-reactivity in biological matrixes

The binding affinity of an antibody does not correlate with biological response for a toxin group. DNA and RNA aptamers are susceptible to enzymatic degradation by nucleases thus requiring highly pure environments. This can be overcome.

Peptide aptamers are constrained to the scaffold protein so they are less flexible which may affect their effectiveness. Only the variable range is used for binding.

  analytical methodologies whereby conversion factors are required especially when certain toxin standards are limited or unavailable for all detectable PSP toxins in a contaminated sample.

  have been developed for the determination of PSP toxins. The method of Lawrence has now been AOAC accredited and accepted following rigorous validation studies by the EU as a first action method for the toxins for which it is validated (AOAC 2005b). However, HPLC methods are slow, labour intensive and analytical standards are difficult to obtain. Oxidation of PSP toxins is required for HPLC with fluorescence detection because they lack a chromophore. For the AOAC HPLC method two oxidation steps (peroxide and periodate) are required resulting in two analytical runs. Lack of standard material, lengthy purification detectability has inhibited the progress in the development of other techniques such as mass spectrometry for routine monitoring. Though advances in mass spectrometry are being made through the use of a zwitterionic hydrophilic interaction chromatography column

  sourcing a suitable competitor to PSP toxins maybe difficult. The limited availability of the saxiphillin receptor to only a few research groups has inhibited the characterization in the role of PSP toxin detection by SPR. Research is on-going in the synthesis of cloned receptors which would prove beneficial in both functional assays and as more stable alternative binders to antibodies for sensor technologies.

  a reduction tool to the number of animal assays performed for regulatory purposes.

  being investigated that could improve the overall toxicity determination of PSP toxins in a sample.

  corresponding non-hydroxylated toxins as in agreement with the ELISA study.

  . They are artificially created ligand binding sites that are able to selectively recognise a target molecule. The synthesis involves the polymerisation of functional and cross-linking monomers around the target of interest or a structural mimic template through noncovalent interactions or reversible covalent bonds or both with the functional groups of the template. These three dimensional polymer based systems can be optimised to yield recognition sites highly specific and selective to the target of interest in the same

  whole PSP toxin family or individual binders for each of the varying toxic groups within this family.

  Usleber E, Dietrich R, Märtlbauer E. 1995. Production and characterisation of antibodies against neosaxitoxin utilizing a novel immunogen synthesis procedure. Food Agr Immunol. 7: 315-322. Campas C, Prieto-Simon B, Marty JL, 2007. Biosensors to detect marine toxins: assessing seafood safety. Talanta 72: 884-895 Campbell, K.; Stewart, L.D; Fodey, T.L.; Haughey, S.A., Doucette, G.J., Kawatsu, K., Elliott, C.T. 2007. Assessment of specific binding proteins suitable for the detection of paralytic shellfish poisons using optical biosensor technology. Anal. Chem. 79: 5906-5914 Campbell K, Huet AC, Charlier C, Higgins C, Delahaut P, Elliott CT. 2009. Comparison of ELISA and SPR biosensor technology for the detection of paralytic shellfish poisoning toxins J Chrom B, 877: 4079-4089 Campbell K, Haughey SA, van den Top H, van Egmond H, Vilarino N, Botana L, Elliott CT. 2010. Single laboratory validation of a surface plasmon resonance biosensor screening method for paralytic shellfish poisoning toxins. Anal. Chem. 82 (7Bürk C, Dietrich R, Elliott CT. (Unpublished data). Use of two antibodies to enhance cross-reactivity for paralytic shellfish poisoning toxins using optical biosensor technology. Carlson RE, Lever ML, Lee BW, Guire PE. 1984. Development of immunoassays for paralytic shellfish poisoning. A radio-immunoassay for saxitoxin. In Seafood Toxins; Ragelis, E.P., Ed; American Chemical Society: Washington, DC, 181-192. Cembella A, Parent Y, Jones D, Lamoureux G. 1990. Specificity and cross-reactivity of an absorption inhibition enzyme linked immunoassay for the detection of paralytic shellfish toxins. In Toxic Marine Phytoplankton; Granéli, E., Sundstrom, B., Edler, L. and Anderson, D.M., Eds.; Elsevier: New York. pp339-344. Cestele S, Catterall WA. 2000. Molecular mechanisms of neurotoxin action on voltagegated sodium channels. Biochimie, 82: 883-892 Chen H, Kim YS, Keum SR, Kim SH, Choi HJ, Lee J. An WG, Koh K. 2007. Surface plasmon spectroscopic detection of saxitoxin. Sensors. 7: 1216-1223. Chianella I, Piletsky SA, Tothill IE, Chen B, Turner APF. 2003. MIP based solid phase extraction cartridges combined with MIP based sensors for the detection of microcystin-LR. Biosens. Biolectron. 18:119-127.

  Luckas B. 2008. Determination of marine biotoxins relevant for regulations: from the mouse bioassay to coupled LC-MS methods. Anal. Bioanal. Chem. 391: 117-134. Chu FS, Fan TSL. 1985. Indirect enzyme-linked immunosorbent assay for saxitoxin in shellfish. J AOAC Int. 68: 13-16. Chu FS, Huang X, Hall S. 1992. Production and characterisation of antibodies against neosaxitoxin. J AOAC Int. 75: 341-345. Chu FS, Hsu KH, Huang X, Barrett R, Allison C. 1996 Screening of paralytic shellfish poisoning toxins in naturally contaminated samples with three different direct competititve enzyme linked immunosorbent assays. J. Agric. Food Chem. 44: 4043-4047. Deeds JR, Landsberg JH, Etheridge SM, Pitcher GC, Longan SW. 2008. Non-traditional vectors for paralytic shellfish poisoning. Mar. Drugs. 6: 308-348 De Jong LAA, Uges DRA, Franke JP, Bischoff R. 2005. Receptor ligand binding assays: Technologies and applications. J. Chrom. B. 829:1-25.

  European Food Safety Authority. Scientific Opinion of the Panel on Contaminants in the Food Chain on a request from the European Commission on Marine Biotoxins in Shellfish-Saxitoxin Group. The EFSA Journal 2009, 1019, 1-76. Fonfria, E.S.; Vilarino, N.; Campbell, K.; Elliott, C.; Haughey, S.A.; Ben-Gigirey, B.; Vieites, J.M.; Kawatsu, K.; Botana, L.M. 2007. Paralytic shellfish poisoning detection by surface plasmon resonance-based biosensors in shellfish matrices. Anal. Chem. 79: 6303 -6311. Gawley RE, Mao H, Haque M, Thorne JB, Pharr JS. 2007. Visible Fluorescence chemosensor for saxitoxin. J. Org. Chem. 72 (6): 2187-2191 Gawley RE, Pinet S, Cardona CM, Datta PK, Ren T, Guida WC, Nydick J, Leblanc RM. 2002. Chemosensors for the marine toxin saxitoxin. JACS. 124(45):13448-13453 Garthwaite I, Ross KM, Miles CO, Briggs LR, Towers NR, Borrell T, Busby P. 2001. Integrated Enzyme-Linked Immunosorbent Assay Screening System for Amnesic, Neurotoxic, Diarrhetic, and Paralytic Shellfish Poisoning Toxins Found in New Zealand. J AOAC Int. 84(5): 1643-1648.

Figure 1 :

 1 Figure 1: The structure, relative molecular mass (RMM) and toxicity factor of paralytic

  Figure 2:

Table 1 :

 1 General advantages and disadvantages of binders previously assessed for SPR analysis of PSP toxins

Table 2 :

 2 Sensitivity and specificity data for both STX and NEO antibody on a NEO chip surface (Campbell et al, unpublished data).

Table 3 :

 3 Sensitivity and specificity of the STX/NEO antibody mix for each PSP toxin (Campbell et al, unpublished data).

Table 4 :

 4 Advantages and disadvantages of potential binders for SPR analysis of PSP

toxins Page 40 of 47 http://mc.manuscriptcentral.com/tfac Email: fac@tandf.co.uk Food Additives and Contaminants F o r P e e r R e v i e w O n l y

		Figure 1:
							R 4
						R 1	H N
							N
			F o		+ H 2 N	N	N H		NH 2 +
	R1	R2	R3	r P Carbamate Toxins R4: OCONH2 RMM Toxicity Factor e e r	OH N-Sulfocarbamoyl toxins OH R 3 R 2 R4: OCONHSO3 -RMM Toxicity Factor	Decarbamoyl toxins R4: OH RMM Toxicity Factor	Deoxydecarbamoyl toxins Factor R4: H RMM Toxicity
	H H H OH OH OH	H H OSO3 -H H OSO3 -	H OSO3 -H H OSO3 -H	STX GTX 2 GTX 3 NEO GTX 1 GTX 4	395.4 395.4 315.3 411.4 411.4	1.00 0.3382 0.6005 1.0911 0.8993 0.6569 R e B1 (GTX 5) C1 C2 B2 (GTX 6) C3 C4 v i e w	379.4 475.4 475.4 395.4 491.4 491.4	0.0632 0.0047 0.0754 0.0607 0.0101 0.0436	dc-STX dc-GTX 2 dc-GTX 3 dc-NEO dc-GTX 1 dc-GTX 4	256.3 352.3 352.3 272.3 368.3 368.3	0.7451 0.1625 0.3978 0.7013 ND ND	do-STX do-GTX 2 do-GTX 3	240.3 336.3 336.3	ND ND ND
							O n
								l y

Table 1 : Page 43 of 47 http://mc.manuscriptcentral.com/tfac Email: fac@tandf.co.uk Food Additives and Contaminants

 1

	Binder	Advantages	Disadvantages
		F o
		r
		P
		e e r
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Table 2 :

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			Food Additives and Contaminants
	Chip surface				Neosaxitoxin Chip Surface
	Antibody			STX			NEO
	Antibody Dilution in HBS-EP			1/250			1/50
	Antibody ratio to standard			1:1			1:1
	Flow rate (µ µ µ µL/min)			25			25
	Contact time (min)			2			2
	PSP Toxin Analogue	IC50 (ng/ml)	% Cross-reactivity	Dynamic range IC20 to IC80 (ng/mL)	IC50 (ng/ml)	% Cross-reactivity	Dynamic range IC20 to IC80 (ng/mL)
	Saxitoxin Dihydrochloride F Neosaxitoxin o Gonyautoxin 1/4		4.8 13.4 474 **	100 35.8 1.0 **	2.7 -8.1 3.7 -84.8 77 -2780 **	41.8 2.4 4.1	100 1742 1032	6.6 -203 1.1 -5.5 1.6 -14.9
	r Gonyautoxin 2/3	7.0	68.7	2.1 -22.0	19.6	213	4.1 -91.6
	Decarbamoyl saxitoxin Decarbamoyl neosaxitoxin P 2.9 100 Decarbamoyl gonyautoxin 2/3 18.7 C1/C2 14.8 e Gonyautoxin 5 4.1 e 165.1 4.8 25.8 32.6 117 r C3/C4 206 ** 2.3 **	1.3 -6.3 14.4 -200 4.4 -62.3 3.3 -53.6 2.7 -7.7 27 -590 **	>1000 192.1 >1000 >1000 >1000 100	<4.2 21.8 <4.2 <4.2 <4.2 41.3	ND 41.9 -512 ND ND ND ND
				R e v i e w
						O n
							l y

://mc.manuscriptcentral.com/tfac Email: fac@tandf.co.uk *ND means not determined due to lack of standard. **Values

calculated based on extrapolation of the 4 parameter fit applied to the standard curve due to availability of standard.

Table 3 :

 3 Page 45 of 47

	Food Additives and Contaminants

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Table 4 :

 4

	Antibody Dilution in HBS-EP		STX NEO			1/500 1/50
	Antibody Mix			20% STX / 80% NEO
	Antibody ratio to standard			1:1
	Flow rate (µ µ µ µL/min)			12
	Injection Volume (µ µ µ µL)			60
	Contact time (min)			5
			Toxin Concentration	Toxin Concentration as STXdiHCl
			(ng/ml)			equivalents (ng/ml)
	PSP Toxin Analogue	IC50 (ng/ml)	% Cross-reactivity	Dynamic range IC20 to IC80 (ng/mL)	IC50 (ng/ml)	% Cross-reactivity	Dynamic range IC20 to IC80 (ng/mL)
	Saxitoxin Dihydrochloride	2.8	100	0.2 -68.8	2.8	100	0.20 -68.8
	Neosaxitoxin	2.8	100	0.8 -9.9	3.1	90.3	0.87 -10.8
	Gonyautoxin 1/4	4.4	63.6	0.8 -25.3	4.0	70	0.72 -22.8
	Gonyautoxin 2/3	4.9	57.1	0.9 -28.0	2.9	96.6	0.54 -16.8
	Decarbamoyl saxitoxin	0.4	700	0.1 -1.2	0.3	933	0.07 -0.9
	Decarbamoyl neosaxitoxin	9.1	30.7	1.7 -48.7	6.4	43.8	1.19 -34.2
	Decarbamoyl gonyautoxin 2/3	17.1	16.4	3.1 -94.7	6.8	41.1	1.23 -37.7
	C1/C2	14.5	19.3	2.0 -102.7	1.1	255	0.15 -7.7
	Gonyautoxin 5	0.4	700	0.1 -1.5	0.03	9333	0.01 -0.1
	C3/C4	91.8	3.0	24.0 -198	4.0	70	1.03 -8.5

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Acknowledgements

This study was funded by the European Commission as part of the 6 th Framework Programme Integrated Project BioCop (contract FOOD-CT-2004-06988) and 7 th Framework Project Conffidence (contract 211326).