Table IV.
SPEAD Titer Calculation (SPEAD Indicates Solid-Phase Extraction with Acid Dissociation)
| Step | Description | Step dilution | Cumulative dilution | |
|---|---|---|---|---|
| Day 1 | Sample predilution | 1/4 dilution of sample | 1/4 | 1/4 |
| Sample pretreatment 1 | Biotin-drug complex: equal volume of sample and biotin-drug | 1/2 | 1/8 | |
| Day 2 | Sample pretreatment 2 | Streptavidin: biotin-drug complex capture (100 µL of prior step) | No dilution | 1/8 |
| Acid dissociation (60 µL of acid (2/1); concentrated back to 1/4; 50 µL transferred) | 1.67/1 | 1/4.8 | ||
| Sample neutralization: 150 µL of base added to 50 µL of acid from prior step | 1/4 | 1/19.2 | ||
| Sample analysis | Coat neutralized sample, block, enzyme-HRP drug, substrate | No dilution | 1/19.2 |
For samples diluted further, add the applicable fold dilution for titer reporting, for example, 1/19.2, 1/38.4, and 1/76.8, if 1/2 scheme is followed