TABLE 2.
Parameters tested in the Pn ECL assay
| Exptl condition or diluent component | Values or agents tested | Recommended value or agenta |
|---|---|---|
| Exptl condition | ||
| Antigen concn (ng/spot) | 2.5, 7.5 | 5 |
| Plate blocking agent | BSA, HSA, SuperBlock buffer | BSA |
| Sample incubation time | 30 min, 2 h | 45 min |
| Secondary antibody concn (μg/ml) | 0.5, 2.0 | 2.0 |
| Secondary antibody incubation time | 30 min, 2 h | 1 h |
| Diluent componentb | ||
| PBS (M NaCl) | 0.3, 0.15 | 0.15 |
| Tween 20 (%) | 0.5, 0.05 | 0.05 |
| Triton X-100 (%) | 0, 0.05 | 0 |
| EDTA | 0%, 5 mM | 0% |
a
For the experimental antigen concentrations, sample incubation times, and secondary antibody incubation times, there was no marked advantage among the factor levels evaluated in the experiment, and therefore, an intermediate level was recommended for subsequent assay development.
b
For the diluent component, assay conditions were tested on a separate DOE with the goal of reducing reactivity in the assay background wells (i.e., serum plus labeled secondary antibody in the absence of PnPs coating) associated with each well.