Table 6. Limits of detection and quantification of epsilon toxin spiked in different ovine intestinal contents.
| Sequential O/N PeTX7/PeTX6-AChE | ||||
|---|---|---|---|---|
| 1/2 diluted intestinal sample | 1/6 diluted intestinal sample | |||
| Sample | LoD (pg/mL) | LoQ (pg/mL) | LoD (pg/mL) | LoQ (pg/mL) |
| Jejunum T325 | 25.5 | 73.1 | 9.0 | 24.2 |
| Jejunum T203 | 45.8 | 140.6 | 26.2 | 80.6 |
| Jejunum 111136 | 14.2 | 40.5 | 4.8 | 11.9 |
| Jejunum S216 | 11.9 | 30.4 | 6.5 | 17.0 |
| Jejunum S218 | 21.7 | 66.6 | 6.1 | 17.8 |
| Jejunum S254 | 26.7 | 79.8 | 7.0 | 20.9 |
| Jejunum S263 | 6.6 | 21.8 | 6.0 | 34.8 |
| Jejunum T180 | 49.1 | 136.3 | 21.2 | 62.9 |
| Jejunum T336 | 16.5 | 54.8 | 6.2 | 19.7 |
| Jejunum T346 | 10.6 | 30.8 | 7.5 | 23.4 |
| Jejunum T349 | 98.7 | 282.0 | 18.0 | 53.2 |
| Jejunum 130010 | 610.0 | N.C. | 4.8 | 13.6 |
| Ileum 130010 | 162.6 | 383.8 | 6.3 | 17.1 |
| Cecum 130010 | 2.4 | 6.9 | 5.9 | 17.6 |
| Colon 130010 | 5.3 | 14.9 | 4.1 | 11.5 |
| Rectum 130010 | 3.3 | 7.7 | 3.9 | 10.5 |
| Jejunum 130033 | 18.1 | 62.1 | 5.9 | 23.1 |
| Ileum 130033 | 29.8 | 93.1 | 12.6 | 39.3 |
| Cecum 130033 | 3.0 | 5.8 | 3.1 | 6.8 |
| Colon 130033 | 3.0 | 8.2 | 2.6 | 7.1 |
| Rectum 130033 | 2.8 | 7.2 | 2.7 | 5.4 |
| Dilution buffer | 7.4 ± 3.6 (n = 11) | 21.8 ± 11.2 (n = 11) | ||
Serial dilutions of epsilon toxin were performed in different matrices: either in dilution buffer (PBS containing 25% fetal calf serum and 0.05% Tween20) or in different clarified ovine intestinal contents diluted 2-fold or 6-fold in dilution buffer. All these dilutions were measured with the overnight sequential enzyme immunoassay PεTX7/PεTX6-AChE (see Materials and methods).
Theoretical limits of detection (LoD = mean of nonspecific binding + 3 standard deviations) and quantification (LoQ = mean of nonspecific binding + 10 standard deviations) were calculated using GraphPad Prism software with a nonlinear regression model using a two-site binding saturation curve fit (total and nonspecific binding).
N.C., not calculable.