The plasma proteome holds great significance in tracking the development, progression and therapeutic response of autoimmune diseases. However, current methodologies lack the sensitivity and dynamic range to measure the diverse array and abundance of proteins in blood. NULISA™, Nucleic acid-linked immunosandwich assay, integrates proximity ligation-based specificity with background suppression and immunocomplex purification using a dual capture and release mechanism to achieve attomolar sensitivity and 12 log dynamic range of detection for multiplexed proteomic analysis. Here, we report the analytical validation of fully automated NULISA on ARGO HT™ with the NULISAseq Inflammation Panel 250.

Limit of detection and dynamic range were determined for each assay from standard curves generated by serial dilution of pooled recombinant proteins. Intra- and inter-assay precision and reproducibility were evaluated across instruments and with different lots of conjugated antibodies for the inflammation panel. Dilutional linearity of each assay was evaluated using a pooled plasma sample. Assay specificity was evaluated by testing for cross reactivity both within the panel of targeted proteins and to untargeted proteins with high homology to targeted proteins. Assay sensitivity was further assessed for target detectability in plasma.

The NULISA Inflammation Panel demonstrated a 12-log dynamic range spanning low abundance targets such as IFNL1 at attomolar concentrations and higher abundance targets like CRP at micromolar concentrations without sample dilution or enrichment. The median intra- and inter-plate CV across the panel were <10%, and the median total CV including different reagent lots was <15%. Dilutional linearity studies demonstrated excellent relative quantitation linearity for 97% of the targets. Cross reactivity studies demonstrated high specificity with <1% cross reactivity for 95% of the targets in the panel. Overall detectability in plasma samples from healthy and diseased donors was over 95%.

Overall, the fully automated NULISA on the ARGO HT platform demonstrated high analytical performance and enables both single-plex and multiplexed detection of a wide dynamic range of proteins in blood for discovery research and validation studies.