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Open Access Technical advance

Optimization of total protein and activity assays for the detection of MMP-12 in induced human sputum

Peter LaPan1*, Jeff Brady2, Christal Grierson2, Margaret Fleming3, Doug Miller4, Joe Sypek3 and Bin Fu1

Author Affiliations

1 Pfizer Global Research and Development, Department of Biological Technologies, 35 Cambridge Park Drive, Cambridge, MA, 02140, USA

2 Translational Medicine Research Collaboration Laboratory, James Arrot Drive, Ninewells Hospital, Dundee, DD1 9SY, UK

3 Pfizer Global Research and Development, Department of Inflammation, 200 CambridgePark Drive, Cambridge, MA, 02140, USA

4 Pfizer Global Research and Development, Department of Discovery Translational Medicine, 500 Arcola Road, Collegeville PA, 19426, USA

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BMC Pulmonary Medicine 2010, 10:40  doi:10.1186/1471-2466-10-40

Published: 2 August 2010

Abstract

Background

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. 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.

Methods

Commercial 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.

Results

Total 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.

Conclusion

Sensitive 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.