Abstract
The protocol describes a novel, rapid, and no-wash one-step immunoassay for highly sensitive and direct detection of the complexes between matrix metalloproteinases (MMPs) and their tissue inhibitor of metalloproteinases (TIMPs) based on AlphaLISA® technology. We describe two procedures: (i) one approach is used to analyze MMP-9–TIMP-1 interactions using recombinant human MMP-9 with its corresponding recombinant human TIMP-1 inhibitor and (ii) the second approach is used to analyze native or endogenous MMP-9–TIMP-1 protein interactions in samples of human plasma. Evaluating native MMP-9–TIMP-1 complexes using this approach avoids the use of indirect calculations of the MMP-9/TIMP-1 ratio for which independent MMP-9 and TIMP-1 quantifications by two conventional ELISAs are needed. The MMP-9–TIMP-1 AlphaLISA® assay is quick, highly simplified, and cost-effective and can be completed in less than 3 h. Moreover, the assay has great potential for use in basic and preclinical research as it allows direct determination of native MMP-9–TIMP-1 complexes in circulating blood as biofluid.
Highlights
Matrix metalloproteinases (MMPs) belong to a large family of zinc-dependent endopeptidases that contribute to tissue remodeling by degrading extracellular matrix
MMP-9 activity is tightly regulated at three different levels, such as activation, synthesis, and inhibition: its synthesis is regulated by adhesion molecules such as integrins, cytokines, and growth factors [9]; similar to other MMPs, MMP-9 is synthesized as a pro-enzyme and is activated by proteolytic cleavage of its propeptide domain by various proteases including plasmin, meprins, furins, and other activated MMPs [10]; it is inhibited by its endogenous inhibitor, tissue inhibitor of metalloproteinases 1 (TIMP-1), which inhibits active MMP-9 by interacting with its catalytic domain in a 1:1 (MMP-9:TIMP-1) stoichiometry [3]
The activity of MMP-9 is controlled by TIMP-1, and both members of the MMP-9–TIMP-1 tandem are implicated in several disease states including cardiovascular and renal disease
Summary
Matrix metalloproteinases (MMPs) belong to a large family of zinc-dependent endopeptidases that contribute to tissue remodeling by degrading extracellular matrix. Solid-phase immunoassays such as the ELISA are the gold standard analytical platform used in preclinical laboratories for quantification of analytes in plasma, including MMP-9 and TIMP-1 While they provide high sensitivity and specificity, these conventional ELISA platforms used for estimating MMP-9–TIMP-1 protein interaction have several inherent limitations:. – Anti-MMP-9 AlphaLISA® Acceptor Beads mix: dilute the AlphaLISA acceptor beads stock solution 100-fold in AlphaLISA assay buffer to obtain a final concentration of 10 μg/mL. – For recombinant human MMP-9 and TIMP-1 immunoassay: ⚬⚬ TIMP-1, 1 mg/mL stock solution: reconstitute the lyophilized protein of 10 μg in 10 μL of Milli-Q water to obtain a final concentration of 1 mg/mL. ⚬⚬ MMP-9, 0.1 mg/mL stock solution: reconstitute the lyophilized protein of 5 μg in 50 μL of Milli-Q water to obtain a final concentration of 0.1 mg/mL. 2300 EnSpireTM Reader Use the plate reader with the following AlphaLISA® standard protocol settings: excitation filter wavelength at 680 nm, emission filter wavelength at 615 nm, measurement time 200 ms, excitation time 80 ms (40%), and the distance between plate and detector 0.2 mm
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