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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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A comparison of commercially available MMP-12 antibodies as capture antibodies in the FRET activity assay format.
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Figure 1: A comparison of commercially available MMP-12 antibodies as capture antibodies in the FRET activity assay format.

Mentions: MMP-12 is the least abundant of human MMP's in sputum, being present at 1/1000 fold the concentration of the more abundant MMPs such as MMP-8 and MMP-9 (data not shown). The specificity of an assay that depended on the intersection of the specificity of the antibody and the FRET peptide would not work for MMP-12, given that any cross reactivity of the antibody with other MMPs, in particular MMP-8 and MMP-9, would saturate the capture antibody binding sites on the plate and reduce sensitivity for MMP-12. We decided that the specificity of the assay must be conferred by the antibody, allowing for the use of a promiscuous FRET peptide. Given this strategy, we screened a panel of antibodies looking at the 24 hour endpoint signal (Figure 1) using 20 μM Substrate V and 10 ng/mL mature MMP-12. Antibodies were coated at either 1 or 10 μg/mL. The highest signal was seen with antibodies from Dendritics (cat# DDX0282) Genway (cat# 15-288-066A) and Abcam (cat# 11614). These antibodies were further tested for specificity against a panel of mature MMPs.


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 comparison of commercially available MMP-12 antibodies as capture antibodies in the FRET activity assay format.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 1: A comparison of commercially available MMP-12 antibodies as capture antibodies in the FRET activity assay format.
Mentions: MMP-12 is the least abundant of human MMP's in sputum, being present at 1/1000 fold the concentration of the more abundant MMPs such as MMP-8 and MMP-9 (data not shown). The specificity of an assay that depended on the intersection of the specificity of the antibody and the FRET peptide would not work for MMP-12, given that any cross reactivity of the antibody with other MMPs, in particular MMP-8 and MMP-9, would saturate the capture antibody binding sites on the plate and reduce sensitivity for MMP-12. We decided that the specificity of the assay must be conferred by the antibody, allowing for the use of a promiscuous FRET peptide. Given this strategy, we screened a panel of antibodies looking at the 24 hour endpoint signal (Figure 1) using 20 μM Substrate V and 10 ng/mL mature MMP-12. Antibodies were coated at either 1 or 10 μg/mL. The highest signal was seen with antibodies from Dendritics (cat# DDX0282) Genway (cat# 15-288-066A) and Abcam (cat# 11614). These antibodies were further tested for specificity against a panel of mature MMPs.

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