Table 1. Analytical comparison of the NIF with the conventional immobilization format (CIF) and covalent immobilization format (CovIF)-based5,9 sandwich ELISAs for the detection of HFA.
| NIF1 | CovIF2 | CIF3 | |
|---|---|---|---|
| Time required for antibody immobilization (h) | 0.5 | 2.5 | 14 |
| Assay duration (h) | ~4 | ~6 | ~20 |
| Detection range (pg mL−1) | 4.9–20,000 | 9.8–20,000 | 151–20,000 |
| LOD (pg mL−1) | 7 | 12 | 226 |
| Analytical sensitivity (pg mL−1) | 10 | 30 | 510 |
| EC50 (ng mL−1) | 2.6 | 3.4 | 5.8 |
| % CV | |||
| Intra-day (n = 5) | 1.2–8.5 | 2.4–10.4 | 4.7–17.4 |
| Inter-day (n = 5) | 2.1–10.2 | 1.7–17.6 | 3.6–20.0 |
| Assays on various substrates | Yes | Yes | No |
| Requirement for crosslinkers | No | Yes | No |
1NIF: new immobilization format by diluting antibody in APTES followed by physical adsorption on the microtiter plate (MTP).
2CovIF: covalent immobilization format by covalent crosslinking of antibody on an APTES modified MTP surface with EDC.
3CIF: conventional immobilization format by physical adsorption of antibody on the MTP.