Fig. 2: Development of an electrochemiluminescent-based assay for the detection of inflammasome-related protein NLRP3.

Anti-NLRP3 antibodies were tested in different capture and detection antibody pairing orientations in 96-well electrode-containing immunosorbent plates (Meso Scale Diagnostics (MSD), Rockville, MD) for their ability to detect NLRP3 recombinant protein. Once an antibody pair was found, the capture antibody was biotinylated and assay conditions were optimized using streptavidin-coated 96-well electrode-containing immunosorbent plates. a The antibody pair identified was able to detect recombinant NLRP3 protein in 1% BSA (r² = 0.9894) down to a concentration of 0.78 ng/mL. R² was calculated using linear regression. b NLRP3 detection was confirmed through immunoprecipitation (IP), where the capture antibody was used as the pull-down antibody and the detection antibody was used as the immunoblotting antibody. c Clean plasma or plasma spiked with 12.5 ng/mL of recombinant NLRP3 protein was run on the optimized electrochemiluminescent assay using biotinylated mouse IgG or biotinylated Cryo 2 as the capture antibody in order to ensure antibody-pair specificity and low non-specific binding. d NLRP3 protein concentration was plotted against electrochemiluminescent read-out (Arbitrary Light Units) for the representative standard curve and all samples analyzed. Axes are on the logarithmic scale. Samples classified as “non-detectable” were imputed by taking the minimum positive value divided by 2. Error bars in a and c represent s.e.m.