Detection of subpicomolar concentrations of human matrix metalloproteinase-2 by an optical biosensor☆
Section snippets
Surface plasmon resonance measurements
Binding experiments were done with the SPR-based instrument Biacore 2000 and sensor chips CM3 and CM5 using the control software version 2.1 and evaluation software version 3.0 (Biacore AB, Uppsala, Sweden). CM3 chips were used for the one-step sandwich assays, CM5 chips were used for ligand characterization.
The running buffer contained 7.8 mM NaH2PO4, 8 mM Na2HPO4, 137 mM NaCl, 0.1 mM CaCl2, 3 mM KCl, 1.5 mM KH2PO4 and 0.02% (v/v) Tween 20, pH 7.2 [4]. This buffer was also used for sample dilution.
Selection and characterization of two ligands for the development of a MMP-2 sandwich assay
To build up a sandwich assay for MMP-2 it was necessary to find two ligands recognizing different sites on the MMP-2 molecule. Three ligands were investigated with Biacore 2000 in detail—gelatine (substrate), anti-MMP-2 antibody Ab-2, and TIMP-2 (inhibitor). Binding of the ligands to MMP-2 was tested in two formats, with immobilized ligand proteins (Table 2, Fig. 1) and in the reverse format with immobilized MMP-2 (Table 3, Fig. 2). The binding response was read 10 s after the beginning of the
Discussion
A highly sensitive assay has been developed by combination of SPR with a particle-enhanced sandwich assay which allows the detection of subpicomolar concentrations of human MMP-2.
Using the proform of MMP-2 for calibration, the lower detection limit was 0.5 pM MMP-2. Since both TIMP-2 [4], [5] and the commercially available antibody Ab-2 bind both the proform and the active form of MMP-2, the assay should be applicable for the determination of both forms of MMP-2.
While optimizing the assay
Acknowledgements
The authors thank Dr. Thorsten Stroh for helpful discussions.
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Financial support by the Federal Ministry of Education and Research, Germany (InnoRegio-BioHyTec Project No. 03i1304C) is gratefully acknowledged.