718 K. Campbell et al. 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 an STX surface was employed, which can be regenerated over 1000 times for reuse 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 non-hydroxylated and hydroxylated toxins indicating that when samples are analysed and compared with the mouse bioassay using this binder the toxin content of a sample would be underestimated if the toxin composition is predomi- nately 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 over- estimate compared with the mouse bioassay. For regulatory purposes, underestimation could have severe health implications to the consumer whereby contaminated material is declared safe for consump- tion, whereas overestimation could cause detrimental economic losses to the industry through the unneces- sary 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 et al. applied different extraction procedures, previously employed for PSP toxin analysis, to different shellfish species and they highlighted that by either using an ethanolic extraction procedure reported by Garthwaite et al. (2001) or the AOAC HPLC acetic acid extraction (AOAC 2005b), 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 com- pared with 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 (Marchesini et al. 2009). The R895 binder showed a much more diverse but narrower pattern in cross-reactivity profile with improved sensitivities to all toxins compared with GT-13A, but the hydroxylated toxins dcNEO and GTX1/4 will require further improvement. Rawn et al. (2009) performed an evaluation of both binders by SPR using the Garthwaite extraction procedure for analysing 88 natural samples and compared the results with the AOAC HPLC method (AOAC 2005b). This study demonstrated that when using this extraction procedure, different shellfish matrix effects were con- siderable compared with a buffer curve, and if a buffer curve were used for analysis, this 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. However, it was recommended that improved antibody response to the hydroxylated toxins would be necessary if SPR were to be a replacement for 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 replacement tool but a reduction tool for the number of animal assays performed for regulatory purposes. 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 with 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 of using SPR over all the other methods were simplicity, ease of use and speed of analysis. This new extraction procedure using pH 5 sodium acetate buffer followed by centrifugation followed by dilution and analysis was then applied to the STX antibody and a single laboratory validation of this SPR method performed highly satisfactorily (Campbell et al. 2010). An inter-laboratory study between seven international laboratories is currently underway. Threshold values for the assay have been