Fig 2: Plasma inhibits MPO-mediated bioluminescence.(a) Representative plasma filtrate titration curves in the presence of 500 ng l−1 MPO imaged with L-012 and H2O2. Whole plasma had a broad inhibitory concentration range with an IC50=28 (21–35) p.p.m. v/v. Removing plasma components >100 kDa narrowed the inhibitory concentration range, but only modestly increased the IC50 to 120 (110–140) p.p.m. Plasma components <5 kDa were not significantly different from <100 kDa in terms of inhibiting MPO bioluminescence (nonlinear regression P=0.21). (b) Endogenous antioxidant titration curves equivalent to plasma with 20 μg l−1 MPO imaged at a 0.4% v/v dilution. Ascorbic acid inhibition IC50=36 (31–41) nM, uric acid IC50=150 (120–180) nM in the presence of 80 ng l−1 MPO. The lower black bar spans the physiological plasma reference range of ascorbic acid adjusted by the same dilution factor; the upper black bars span the male and female uric acid reference ranges20. Representative data (n=4) are mean±s.d. Regression 95% confidence bands shown in grey.

Fig 3: MAPS reproducibility.MAPS assays independently replicated four times on 72 clinical plasma samples. Dashed lines show median and one-sided 95th percentile values. Computation details are provided in online methods. (a) Absolute replication variability quantified by accounting for both intra- and inter-replicate uncertainty. Median variability was 0.67 μg l−1 MPO, and 95% of samples were reproducible within 5.8 μg l−1 MPO. Variability was scaled with mean MPO by a factor of 0.12 (0.11–0.13). Dashed lines show mean and two-sided 95% coverage variability estimates. (b) Relative replication variability by normalization to variance-weighted replicate mean MPO. Median coefficient of variability was 8.6% with an upper 95th percentile threshold of 27%. Dashed lines show median and one-sided 95th percentile estimate.

Fig 4: MAPS assay with RBP carrier.(a) Example standard solution image and (b) calibration curve constructed with purified human MPO diluted into RBP for MAPS assay. Data are plotted as mean±s.d. (n=3). Triplicate 95% prediction band shown in grey. (c) MAPS assay results using human plasma from healthy volunteers supplemented with purified human MPO to simulate samples covering the range of a commercial ELISA kit. Data are plotted as regressed value±95% confidence intervals. Dashed line represents 1:1 agreement between supplemented MPO and RBP-regressed MPO.

Fig 5: Halogenation substrates for MPO bioluminescence.(a) As suggested by the literature, MPO utilizes physiological concentrations of NaCl, Km=17 (10–23) mM, less effectively than (b) NaBr, Km=2.3 (1.7–2.9) mM. Although reported as even more efficient substrates for MPO, (c) I− and (d) SCN− do not produce similar MPO-mediated bioluminescence with L-012 under these reaction conditions. Representative data (n=4) are shown as mean±s.d.. Regression 95% confidence bands are shown in grey when applicable. MPO is labelled with final imaging concentration.