Abstract
BackgroundProteolysis of matrix components, in particular elastin, is a major contributing factor to the development of lung diseases such as emphysema and chronic obstructive pulmonary disease (COPD). MMP-12 (macrophage elastase) is a protease known to be involved in the progression of lung disease. The relatively low abundance of MMP-12 has precluded the development of quantitative assays that can accurately measure MMP-12 protein levels and activity across cohorts of healthy and diseased individuals.MethodsCommercial antibodies were screened for performance in sandwich ELISA and capture FRET activity assay formats. Precision, accuracy, sensitivity, dilution linearity, and spike recovery were evaluated using sputum samples.ResultsTotal protein and capture FRET activity assays were developed that were sensitive enough to detect MMP-12 in 37 of 38 donor sputum samples. A comparison of results between the two assays shows that a majority of sputum MMP-12 is in the active form. No differences were seen between normal, asthmatic, and COPD donors.ConclusionSensitive and quantitative assays for both MMP-12 activity and total protein in human induced sputum have been developed. These assays can be used to evaluate MMP-12 as a biomarker for lung disease, and to monitor efficacy of potential therapeutic compounds.
Highlights
Proteolysis of matrix components, in particular elastin, is a major contributing factor to the development of lung diseases such as emphysema and chronic obstructive pulmonary disease (COPD)
Tetrazolium amplification combined with direct alkaline phosphatase conjugation provided enhanced sensitivity, which allowed for a 1:8 dilution of sputum samples
Results in Additional file 1, Table S5 show that the percent difference in calculated matrix metalloproteinase (MMP)-12 concentrations between samples with and without IIR ranged from -139% to 92%, with 27 of 38 samples showing an absolute difference greater than 25%
Summary
Proteolysis of matrix components, in particular elastin, is a major contributing factor to the development of lung diseases such as emphysema and chronic obstructive pulmonary disease (COPD). MMP-12 (macrophage elastase) is a protease known to be involved in the progression of lung disease. MMP-12, know as macrophage elastase, is a member of the matrix metalloproteinases. Active MMP-12 has the capacity to degrade extracellular matrix components, in particular elastin [3], and is involved in matrix remodeling both in normal development and during pathophysiological disease. Disruption of the balance between MMP-12 and its endogenous inhibitors can result in diseases such as emphysema and COPD. The mechanism involves the induction of TNF-alpha release from macrophages followed by endothelial cell activation, neutrophil recruitment and secretion of matrix degrading proteases [5]. The resulting elastin fragments have been shown to chemotactic for monocytes, resulting in further recruitment and amplification of the inflammatory cascade [6]
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