Table 1.
Detection of Cyclic Imine Toxins by the Microplate-Receptor Binding Assay: Binding Inhibition Parameters and Comparative Performance with Radioactive and Nonradioactive Assaysa
| 13,19-didesmethyl spirolide-C | 13-desmethyl spirolide-C | pinnatoxin-G | 20-methyl spirolide-G | gymnodimine-A | pinnatoxin-A | |
|---|---|---|---|---|---|---|
| IC50 (nM ± SEM) M- RBASTD-MeOH |
3.10 ± 1.23 | 6.84 ± 1.15 | 20.05 ± 13.32 | 25.57 ± 10.83 | 74.23 ± 10.80 | 75.82 ± 7.09 |
| IC50 (nM ± SEM) M- RBASTD-matrix |
2.57 ± 1.06 | 7.14 ± 1.07 | 24.74 ± 11.44 | 23.79 ± 11.51 | 70.40 ± 13.23 | 78.29 ± 15.27 |
| Kib (nM ± SEM) | 0.80 ± 1.22 | 1.77 ± 1.14 | 5.20 ± 1.33 | 6.63 ± 1.09 | 19.25 ± 8.28 | 23.00 ± 12.16 |
| LODc (nM/Inh%) | 0.8/15.8 | 1/21.5 | 2/15.5 | 2/10.2 | 2/5.2 | 5/9.8 |
| LOQd (nM/Inh%) | 2.0/34.5 | 4/42.1 | 8/32.5 | 6/21.7 | 20/28.5 | 24/23.4 |
| Hill slope | 1.33 ± 0.23 | 1.09 ± 0.14 | 0.65 ± 0.10 | 0.93 ± 0.06 | 0.82 ± 0.09 | 1.31 ± 0.21 |
| R2 | 0.9831 | 0.9915 | 0.9861 | 0.9988 | 0.9977 | 0.9826 |
| Ki (nM ± SEM) (RL-BA) | – | 0.080 ± 0.00220 | – | – | 0.23 ± 0.0820 | – |
| IC50 (nM ± SEM) (FP) | – | 108.2 ± 11.927 | – | – | 391.0 ± 55.327 | – |
| IC50 (nM ± SEM) (FP) | 63.60 ± 333 | – | – | – | – | |
| IC50 (nM ± SEM) (SP-RA) | – | 31.4 ± 4.934 | – | – | 526.0 ± 38.134 | – |
Abbreviations: M-RBASTD-MeOH, IC50 was determined using methanolic standards; M-RBASTD-matrix, IC50 was determined using methanolic standards in the presence of 25 mg/mL P. maximus extract; RL-BA, radioligand binding assay; FP, fluorescence polarization receptor assay; SP-RA, solid-phase receptor-based assay).
Ki was calculated by nonlinear regression analysis (biotin-α-BgTx = 80 nM; Kd of biotin-α-BgTx = 28.3 nM).
LOD was calculated from mean OD492 nm values at toxin concentrations lower than 100 pM ± 3 times the standard deviation (SD).
LOQ was calculated from mean OD492 nm values at toxin concentrations lower than 100 pM ± 10SD.