Matrix metalloproteinase 2, 8 and 9 measurements.
Total matrix metalloproteinase levels, the sum of active MMP and activatable pro-MMP were quantified with specific immuno-capture activity assays. In the first step of this assay the sample is incubated with a 96-well plate coated with specific antibodies recognizing a specific
MMP. In this study plates coated with specific antibodies recognizing MMP-2, MMP-9, or
MMP-8 were used (2,3). After capture the plates were washed and the activity of captured
MMP (MMP-2, -9 or -8 depending on the anti body used for coating) was determined by addition of a pro-detection enzyme ( in this case a modified pro-urokinase that contains a generic MMP activation sequence) (1). The captured MMP converts the modified prodetection enzyme to an active detection enzyme and t6he resulting activity is measured using a urokinase specific peptide substrate (<Glu-Gly-Arg-pNA, S2444 Chromogenix). Since the latter two steps are running simultaneously, the final assay is a parabolic rate assay in which absorbance changes linearly with the square of the incubation time. The absorbance after a fixed time, or the slope of the absorbance change with time squared is linearly dependent on the MMP concentration in the sample. The assay can be used to determine active MMPs, or latent pro-MMPs. The latter are determined after activation of the captured pro-MMP on the plate by treatment with the organo mercurial APMA, which induces a conformation change of the pro-MMP leading to activity. The MMP-2, -9 and -8 assays were obtained in kit-form from
GE Healthcare LifeSciences, Buckinghamshire, UK, article numbers RPN-2631, RPN-2634 and RPN-2635 respectively. The MMP-8 assay RPN-2634 was recently discontinued by GE
Healthcare, but will be available shortly from QuickZyme Biosciences, Leiden, The
Netherlands (www.QuickZyme.com). Specificity and sensitivity of the MMP-2 and -9 assays can be found in the kit manual. The MMP-8 assay shows 7.9% cross reactivity with MMP-1 and less than 2% cross reactivity with MMP-2, -3, -9 and -13. The sensitivity of the MMP-8 assay depends on the incubation length and can go as low as 0.02 ng/ml after 24 h incubation. The necessary dilution range for linearity was established for the tissue extracts by testing various dilutions.
References
1. Verheijen JH, Nieuwenbroek NME, Beekman B, Hanemaaijer R, Verspaget HW,
Ronday HK, Bakker AHF. Modified proenzymes as artificial substrates for proteolytic enzymes: colorimetric assay of bacterial collagenase and matrix metalloproteinase activity using modified pro-urokinase. Biochem. J.
1997;323, 603-609.
2. Vernooy JHJ, Lindeman JHN, Jacobs JA, Hanemaaijer R, Wouters EFM. Increased activity of matrix metalloproteinase-8 and matrix metalloproteinase-9 in induced sputum from patiens with COPD. Chest 2004;126,1802-1810.
3. van de Borne SWM, Cleutjens JPM, Hanemaaijer R, Creemers EE, Smits JFM,
Daemen MJAP, Blankesteijn WM. Increased matrix metalloproteinase-8 and -9 activity in patients with infarct rupture after myocardial infarction. Cardiovascular
Pathology 2009;18, 37-43.