Detection of subpicomolar concentrations of human matrix metalloproteinase-2 by an optical biosensor.
We describe in this paper the development of a one-step sandwich assay for the highly sensitive and fast detection of human matrix metalloproteinase (MMP)-2 (EC 3.4.24.24), using surface plasmon resonance (SPR). For the assay, two ligands were selected: monoclonal anti-MMP-2 antibody Ab-2 and the tissue inhibitor of metalloproteinases (TIMP)-2. They were chosen on the basis of (1) their affinities to MMP-2, (2) the efficiency of immobilization to the sensor chip, (3) the efficiency of adsorption to colloidal gold, and (4) the stability of these protein-coated gold particles. The assay included mixing of MMP-2 with antibody Ab-2 adsorbed to colloidal gold with a diameter of about 20nm and injection into the flowcell of the SPR instrument containing immobilized TIMP-2. By using colloidal gold particles an amplification factor of 114 and a detection limit of 0.5pM for MMP-2 were obtained. The precision of the assay was high even at low analyte concentrations, the standard deviation being 8.3% for five determinations of 1pM MMP-2. No significant binding was observed with the structurally related MMP-9. The assay is far more sensitive and faster than commonly used methods for MMP-2 detection. As TIMP-bound MMP-2 is not detected by this method, the assay can be applied for measuring free MMP-2, reflecting the imbalance of free and inhibitor-bound enzyme in various pathological situations.
Institute of Biochemistry and Biology, University of Potsdam, Karl-Liebknecht-Str. 24-25, Haus 25, D-14476, Golm, Germany. upieper@rz.uni-potsdam.de
Ursula
U
Pieper-Fürst
Ulrike
U
Kleuser
Walter F M
WF
Stöcklein
Axel
A
Warsinke
Frieder W
FW
Scheller
D000906:Antibodies / Q000276:immunology; D015374:Biosensing Techniques / Q000379:methods*; D003102:Colloids; D006046:Gold / Q000378:metabolism; D006801:Humans; D007700:Kinetics; D008024:Ligands; D020778:Matrix Metalloproteinase 2 / Q000032:analysis* / Q000378:metabolism; D013997:Time Factors; D019716:Tissue Inhibitor of Metalloproteinase-2 / Q000276:immunology / Q000378:metabolism
D000906:Antibodies; D003102:Colloids; D008024:Ligands; D019716:Tissue Inhibitor of Metalloproteinase-2; D006046:Gold; D020778:Matrix Metalloproteinase 2