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


CONCLUSIONS
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.
LaPan
Peter
P
Department of Biological Technologies, Pfizer Global Research and Development, 35 Cambridge Park Drive, Cambridge, MA 02140, USA. plapan@wyeth.com
Brady
Jeff
J
Grierson
Christal
C
Fleming
Margaret
M
Miller
Doug
D
Sypek
Joe
J
Fu
Bin
B
D000918:Antibody Specificity; D002138:Calibration; D004549:Elastin / Q000378:metabolism; D004797:Enzyme-Linked Immunosorbent Assay / Q000379:methods* / Q000592:standards; D031541:Fluorescence Resonance Energy Transfer / Q000379:methods* / Q000592:standards; D006801:Humans; D007201:Indicator Dilution Techniques; D053508:Matrix Metalloproteinase 12 / Q000276:immunology / Q000378:metabolism*; D029424:Pulmonary Disease, Chronic Obstructive / Q000175:diagnosis* / Q000378:metabolism; D012015:Reference Standards; D015203:Reproducibility of Results; D013183:Sputum / Q000201:enzymology*
D004549:Elastin; D053508:Matrix Metalloproteinase 12