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

LaPan P, Brady J, Grierson C, Fleming M, Miller D, Sypek J, Fu B - BMC Pulm Med (2010)

Bottom Line: 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).Precision, accuracy, sensitivity, dilution linearity, and spike recovery were evaluated using sputum samples.No differences were seen between normal, asthmatic, and COPD donors.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Biological Technologies, Pfizer Global Research and Development, 35 Cambridge Park Drive, Cambridge, MA 02140, USA. plapan@wyeth.com

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.

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Related in: MedlinePlus

A pairwise plot of interpolated MMP-12 concentrations for both activity and total MMP-12 protein concentration in normal, COPD, and asthmatic donors.
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Figure 4: A pairwise plot of interpolated MMP-12 concentrations for both activity and total MMP-12 protein concentration in normal, COPD, and asthmatic donors.

Mentions: The MMP-12 total protein and activity assays were used to assess total protein and activity levels in induced sputum from 18 healthy, 10 asthma, and 10 COPD donors. Figure 4 shows that interpolated concentrations for MMP-12 are similar for both total protein and activity, suggesting that a majority of the MMP-12 in sputum is in the mature, active form. A comparison of MMP-12 levels across disease states (Figure 5) shows that the differences do not reach significance at the p = 0.05 level (Student's t-test).


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

LaPan P, Brady J, Grierson C, Fleming M, Miller D, Sypek J, Fu B - BMC Pulm Med (2010)

A pairwise plot of interpolated MMP-12 concentrations for both activity and total MMP-12 protein concentration in normal, COPD, and asthmatic donors.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2921351&req=5

Figure 4: A pairwise plot of interpolated MMP-12 concentrations for both activity and total MMP-12 protein concentration in normal, COPD, and asthmatic donors.
Mentions: The MMP-12 total protein and activity assays were used to assess total protein and activity levels in induced sputum from 18 healthy, 10 asthma, and 10 COPD donors. Figure 4 shows that interpolated concentrations for MMP-12 are similar for both total protein and activity, suggesting that a majority of the MMP-12 in sputum is in the mature, active form. A comparison of MMP-12 levels across disease states (Figure 5) shows that the differences do not reach significance at the p = 0.05 level (Student's t-test).

Bottom Line: 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).Precision, accuracy, sensitivity, dilution linearity, and spike recovery were evaluated using sputum samples.No differences were seen between normal, asthmatic, and COPD donors.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Biological Technologies, Pfizer Global Research and Development, 35 Cambridge Park Drive, Cambridge, MA 02140, USA. plapan@wyeth.com

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.

Show MeSH
Related in: MedlinePlus