its activity. In contrast, acetone preserved MPO activity, and over three different concentrations of MPO, we were able to recover 9665.2% of activity. This validated acetone protein precipitation as a feasible method to concentrate samples that contain MPO. ECF and ICF Protein Extracts Contain Substances that Interfere with MPO Activity Measurements To test if our assays could efficiently recover MPO from biological samples, we performed a spike and recovery experiment, where a known amount of human MPO was added to both ECF and ICF extracts from several organs, and MPO activity was measured thereafter. We selected three different MPO assay methods from the literature, which have all been reported to be sensitive and specific to MPO: 1) Bromide dependent chemiluminescence with luminol at acidic pH, 2) peroxidase activity with ADHP, and 3) chlorination activity with APF and HPF, where the subtraction of HPF signal from APF signal is thought to represent specific MPO activity. Results were normalized as percentage activity of pure enzyme. For both ECF and ICF, MPO recovery was variable and dependent on the assay and organ used, without a clear recognizable trend. Of note, a large range of MPO activity levels was found, which suggested that peroxidases other than MPO and/or other interfering substances were likely affecting the three assay methods. The nonspecificity of these assays was further confirmed by assaying different concentrations of hemoglobin, which has peroxidase activity, with these probes. ADHP and luminol showed a dose-dependent signal increase in these circumstances. Based on these findings we conclude that it is necessary to utilize a more specific method for MPO activity detection and hypothesized that antibody-specific binding or extraction of MPO from biological samples before measuring enzyme activity would likely circumvent these issues. Measuring MPO Activity MPO Antibody Capture Assay is Highly C.I. Natural Yellow 1 biological activity Reproducible To remove interfering substances from the biological samples being tested, we used an antibody capture 11784156 assay. First, to establish the reproducibility and linear range of this assay, we loaded homogenized murine neutrophils into anti-MPO antibodycovered wells, and after binding washed away any interfering substances. We then measured MPO activity with ADHP, chosen for its high sensitivity and assay range. For intra-assay reproducibility, triplicates were run simultaneously, while for inter-assay reproducibility, each standard curve was run at least 1 hour apart. The range of the linear part of the curve was found to be from 598 to 1.2 million neutrophils with coefficients of determination of 0.98 and 0.88 for intra-assay and inter-assay reproducibility, respectively. 6 Measuring MPO Activity This experiment also allowed us to evaluate the sensitivity of this assay, which can detect MPO from as few as 500 neutrophils. MPO Anti-body Capture Assay Increases the Specificity of MPO 11078888 Activity Assays in Different Disease Models Next, we validated the specificity of this assay using three different murine inflammatory disease models: 1) EAE, 2) NASH, and 3) MI. We evaluated brains for EAE, livers for NASH, and hearts and plasma for MI in both WT and MPO-KO mice. Both intra- and extracellular extracts were run on the antibody-capture assay with ADHP, as well as with ADHP, luminol, and APF/HPF without antibody-capture. enates in these three disease models. In fact, without antibody-capture, the measured MPO activity was hi