Limits...
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.

Show MeSH

Related in: MedlinePlus

Vmax (abscissa) is plotted against Anaspec substrate (ordinate) in a solution phase assay using 20 μM substrate and 100 ng/mL pro or autocatalyzed MMP-12.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC2921351&req=5

Figure 3: Vmax (abscissa) is plotted against Anaspec substrate (ordinate) in a solution phase assay using 20 μM substrate and 100 ng/mL pro or autocatalyzed MMP-12.

Mentions: In parallel, catalytic MMP-12 was tested against a panel of FRET substrates. Substrate III provided the greatest Vmax and was used in all subsequent assays (Figure 3).


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)

Vmax (abscissa) is plotted against Anaspec substrate (ordinate) in a solution phase assay using 20 μM substrate and 100 ng/mL pro or autocatalyzed MMP-12.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 3: Vmax (abscissa) is plotted against Anaspec substrate (ordinate) in a solution phase assay using 20 μM substrate and 100 ng/mL pro or autocatalyzed MMP-12.
Mentions: In parallel, catalytic MMP-12 was tested against a panel of FRET substrates. Substrate III provided the greatest Vmax and was used in all subsequent assays (Figure 3).

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