• Minimal non-specific background and strong responses to analyte yield high signal-to-background ratios.
• The stimulation mechanism (electricity) is decoupled from the response (light signal), minimizing matrix interference.
• Only labels bound near the electrode surface are excited, enabling non-washed assays.
• Labels are stable, non-radioactive, and directly conjugated to biological molecules.
• Emission at ~620 nm eliminates problems with color quenching.
• Multiple rounds of label excitation and emission enhance light levels and improve sensitivity.
• A carbon electrode surface has 10X greater binding capacity than polystyrene wells.
• Surface coatings can be customized

The panel was developed using the S-PLEX platform, which uses an enhanced, electrochemiluminescence reporter technology. Critical components such as immunoassay plates, diluents, blockers, labels, and other reagents were optimized to enable the multiplexing of high-sensitivity assays. Protocols were optimized for ease of use, optimal performance, and robustness. Performance characterization testing of the nine assays in this panel included limit of detection (LOD), lower limit of quantitation (LLOQ), upper limit of quantitation (ULOQ), dilution linearity, and spike recovery. Multiple matrices were also interrogated including serum, EDTA/heparin/citrate plasma, CSF, and stimulated cell supernatants. 

Results

Calibration Curves, Assay Ranges

The assays were analytically tested using 25 μL sample volumes per well to assess all analytes simultaneously. Of the 9 assays analyzed, 3 had LLODs below 10 fg/mL, 3 had LLODs below 20 fg/mL, and the remaining assays were below 70 fg/mL with dynamic ranges of at least 3 logs and intraplate %CVs <10%. Measurements shown are adjusted to NIBSC/WHO standards.

Cross-reactivity between the assays was less than 0.5% in all cases.

Conclusions