Determination of Gelatinase-B (MMP-9) activity using a novel immunocapture assay in comparison with specific elisas

Gelatinase-B has been shown to be produced by a variety of different cells, fibroblasts in particular. It has also been shown to be elevated in the sera of patients with specific disease states, for example hepatocellular carcinoma. A novel technology is described for determining the activity of matrix metalloproteinases (MMPs) using a colorimetric assay. The assay is based on a modified pro-urokinase constructed by protein engineering. In the modified prourokinase the activation sequence, normally recognised by plasmin (Pro-Arg-Phe-Lys # Ile-Ile-Gly-Gly), was replaced by a sequence that is specifically recognised by MMPs (Arg-Pro-Leu-Gly # Ile-Ile-Gly-Gly). A chromogenic peptide substrate for urokinase is then used to measure the active urokinase, generated through MMP activation of the modified urokinase. The assay uses a MMP-9 specific monoclonal antibody, coated 96 well microtitre plate to confer MMP-9 specificity. MMP-9 is captured from biological fluids or cell culture media through overnight incubation of the samples at 4°C, in the microtitre plate wells. Immobilised MMPs are then activated using APMA for a 2hour incubation period at 37°C. Differential activation with APMA, enables the analysis of the activity of both the active and latent forms of MMP-9. Detection incubation with modified urokinase and the substrate S-2444 continues at 37°C for 2 to 6 hours. The normal assay range is between 32 and 2ng/ml for a 2 hour incubation. A more sensitive assay range between 8 and 0.5ng/ml, enabled a detection sensitivity of 0.5ng/ml for a 6 hour detection incubation period. Cell culture and serum/plasma samples were used to validate the activity assay. U937 cell cultures, stimulated under different conditions, were assayed in the activity assay and in a variety of MMP-9 specific ELISAs. The APB RPN 2614 ELISA used, specifically detects pro MMP-9. In addition other antibodies were used to construct ELISAs for the measurement of total MMP-9 and pro and active MP-9/TIMP- 1 complexes. Further validation work was performed using serum and a variety of plasma types, for linearity and recovery experiments. It was observed that greater than a 1:16 dilution of these samples was required to obtain a high recovery. Normal latent MMP-9 levels were found to be detectable at this dilution. MMP-9 activity in normal biological samples was detected using the novel activity assay. The activity assay results were shown to correlate with results obtained from the MMP-9 specific ELIS As. A separate study determined the levels of Gelatinase B (MMP-9) activity in saliva samples from patients with Sjögren s syndrome. An assay based on radiolabelled gelatinated collagen showed that gelatinase activity was slightly, although not significantly, increased in Sjögren s syndrome. The novel immunocapture activity assay however, specifically measuring MMP-9, showed that MMP-9 levels were increased in Sjögren s patients compared with healthy controls: MMP-9 (already active): patients 8.9 + 2.5 U/mg, controls 1.0+0.5 U/mg (p=0.002); latent plus active MMP-9: patients 53.1 ±9.8 U/mg, controls 16.5 + 2.6 U/mg (p=0.010). © Warcourt Brace & Co. Ltd 1999.