Food Additives and Contaminants 719 Table 2. Sensitivity and specificity data for both STX and NEO antibody on a NEO chip surface (Campbell et al., unpublished data). Chip surface Antibody Antibody dilution in HBS-EP Antibody ratio to standard Flow rate (ml min 1) Contact time (min) PSP toxin analogue Saxitoxin dihydrochloride Neosaxitoxin Gonyautoxin 1/4 Gonyautoxin 2/3 Decarbamoyl saxitoxin Decarbamoyl neosaxitoxin Decarbamoyl gonyautoxin 2/3 C1/C2 Gonyautoxin 5 C3/C4 4.8 13.4 474b 7.0 2.9 100 18.7 14.8 4.1 206b STX 1/250 1:1 25 2 100 35.8 1.0b 68.7 165.1 4.8 25.8 32.6 117 2.3b Neosaxitoxin chip surface Dynamic range, IC(n20g tmolIC1)80 2.7–8.1 3.7–84.8 77–2780b 2.1–22.0 1.3–6.3 14.4–200 4.4–62.3 3.3–53.6 2.7–7.7 27–590b 41.8 2.4 4.1 19.6 41000 192.1 41000 41000 41000 100 NEO 1/50 1:1 25 2 100 1742 1032 213 54.2 21.8 54.2 54.2 54.2 41.3 Dynamic range, IC20 to IC80 (ng ml 1) 6.6–203 1.1–5.5 1.6–14.9 4.1–91.6 n.d.a 41.9–512 n.d. n.d. n.d. n.d. (ngICm5l0 1) Percentage cross-reactivity (ngICm5l0 1) Percentage cross-reactivity Notes: an.d., Not determined due to lack of the standard. bValues calculated based on extrapolation of the four-parameter fit applied to the standard curve due to availability of the standard. 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 by regulatory monitoring laboratories. To date there has been no reports of PSP toxins occurring in isolation, but if GTX1/4 were 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 formats are being investigated that could improve the overall toxicity determination of PSP toxins in a sample. A combination of two binders, one raised to STX (Campbell et al. 2007), the other to NEO (Bu¨ rk et al. 1995), were assessed on a NEO chip surface to deter- mine 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each in order to be able to detect the full range of PSP toxins in isolation at the regulatory level (Campbell et al., unpublished data).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 hydroxyl- ated toxins at the R1 position respectively (Table 2). The percentage cross-reactivity of STX antibody in relation to STXdiHCl was variable in relation to toxin structure with the NEO surface. Toxins with modifi- cations in the R4 position (Figure 1) displayed the highest percentage cross-reactivity followed by those with modifications to the R2 and R3 positions and then those toxins hydroxylated in the R1 position. Combinations of modifications showed an additive decrease in percentage cross-reactivity with the outcome for GTX1/4, which is modified at the R1, R2 and R3 positions, displaying the lowest percentage 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 (Bu¨ rk et al. 1995). This antibody was significantly more sensitive to the R1 hydroxylated toxins compared with the corresponding non-hydroxylated toxins as in agreement with the ELISA study. 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 percentage 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 10 ng ml 1 (Figure 5). The full cross-reactivity profile was deter- mined 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,