<i>In vivo</i> imaging and <i>in vitro</i> MPO activity assays demonstrate markedly different findings.

<p>(<b>A</b>) MPO-Gd molecular MR imaging reveals MPO inhibition in vivo in mice with experimental autoimmune encephalomyelitis that were treated with ABAH. MPO activity maps are shown in 3D from two angles (left), as well as overlays of MPO activity maps over T1 images (right). (<b>B</b>) Quantification of imaging reveals significant difference in MPO activity <i>in vivo</i> (<i>P</i> = 0.03, n = 8 per group). (<b>C</b>) <i>In vitro</i> assays on whole tissue homogenates using ADHP or TMB do not confirm the <i>in vivo</i> imaging finding (<i>P</i> = 0.68 and 0.88, respectively, n = 4 per group). *: <i>P</i><0.05, n.s. = not statistically significant. MPO = myeloperoxidase. TMB = 3,3′,5,5′-Tetramethylbenzidine. ADHP = 10-acetyl-3,7-dihydroxyphenoxazine. ABAH = 4-aminobenzoic acid hydrazide. Activation ratio = contrast-to-noise ratio 60 minutes over 15 minutes post MPO-Gd injection.</p

Intracellular MPO activity correlates well with tissue neutrophil content.

<p>(<b>A</b>) Flow cytometry demonstrates different neutrophil counts in brain, heart, liver, spleen, and bone marrow, as quantified in (<b>B</b>) (n = 2 per group). (<b>C</b>) Intracellular MPO activity was measured with the antibody-capture assay using ADHP, and shows a similar trend to neutrophil content per organ (n = 2 per group). (<b>D</b>) A close correlation was found between neutrophil content and intracellular MPO activity in these organs. MPO = myeloperoxidase. ADHP = 10-acetyl-3,7-dihydroxyphenoxazine.</p

Antibody capture improves the specificity of MPO activity assays on extra- and intracellular extracts in various models of inflammatory diseases.

<p>(<b>A</b>) Antibody capture of MPO followed by activity detection with ADHP reveals high specificity towards MPO. This is shown in extra- and intracellular fractions in brains from EAE mice, livers from mice with NASH, and hearts and plasma from mice with myocardial infarction (n = 3 per group). (<b>B</b>) The same samples processed without antibody capture reveal poor specificity towards MPO, and no significant difference between WT and MPO-KO mice (n = 3 per group). * <i>P</i><0.05. ** <i>P</i><0.01. *** <i>P</i><0.001. ADHP = 10-acetyl-3,7-dihydroxyphenoxazine. MPO = myeloperoxidase. EAE = experimental autoimmune encephalomyelitis. MI = myocardial infarction. NASH = non-alcoholic steatohepatitis. ECF = extracellular fraction. ICF = intracellular fraction. WT = wildtype C57BL/6. MPO^−/− = MPO knockout.</p

MPO in the literature.

<p>(<b>A</b>) Usage of MPO activity assays in the Literature from 2011 to 2012. (<b>B</b>) Manuscripts published on MPO from 1990 to 2012; grey bars indicate manuscripts considered in (<b>A</b>). MPO = myeloperoxidase. TMB = 3,3′,5,5′-Tetramethylbenzidine. ADHP = 10-acetyl-3,7-dihydroxyphenoxazine. BALF = bronchoalveolar lavage fluid. Ab = antibody. APF = 3′-(p-aminophenyl) fluorescein. ELISA = enzyme-linked immunosorbent assay.</p

Spike and recovery assay: tissue homogenates and extracellular fluid contain interfering substances.

<p>(<b>A</b>) Extracellular protein fraction from different organs contains substances interfering with ADHP, luminol, and APF assays (n = 2 per group). (<b>B</b>) Intracellular protein fractions also contain interfering substances (n = 2 per group). MPO = myeloperoxidase. ADHP = 10-acetyl-3,7-dihydroxyphenoxazine. APF = 3′-(p-aminophenyl) fluorescein. HPF = 3′-(p-hydroxyphenyl) fluorescein.</p

Validation of Extracellular Protein Isolation and MPO Protein Precipitation.

<p>(<b>A</b>) LDH assay of intra- and extracellular protein fractions of different organs shows that the extracellular fraction only contains very low levels of LDH activity, while the intracellular fraction contains the majority of the LDH activity (left). LDH ratio shows a 90 or higher fold level of ICF LDH over ECF LDH activity (right). (<b>B</b>) Protein precipitation of MPO with acetone has no effect on its activity, as evaluated with ADHP (n = 2 per group). LDH = lactate dehydrogenase. BCA = bicinchoninic acid. MPO = myeloperoxidase.</p