H04L 5/003, H04W 28/18, H04W 72/0446, C12Q 1/40, C12Y 302/01, C12Y 302/01004, C12Y 302/01022, C12Y 302/01025, C12Y 302/01055, D21H 11/18, D21H 11/20, G01N 2333/924, G01N 2333/94, G01N 2333/942, G01N 30/64, G01N 30/7233, G01N 30/96, G01N 33/34, G01N 33/343
A method of measuring soluble or insoluble cell or tissue-associated collagenase activity. The substrate includes native collagen fibrils that were stained with COOMASSIE Brilliant Blue R-250. Incubation with collagenase can be observed in real-time by the generation of digested smaller fragments. The degraded blue fragments are obtained by filtration through class fibers, onto which intact collagen fibrils are retained. The filtrate containing the blue collagen fragments is incubated with a detergent in order to extract the blue dye, and the mixture is centrifuged in order to separate the dye in the supernatant from the pellet, which contains de-stained collagen fragments and other insoluble materials contained in the test samples (such as bacterial cells or tissues). The amount of dye extracted is quantified by measuring the amount of dye extracted from these fragments, i.e., the absorbance at 600 nm using a spectrophotometer or an ELISA reader.
Dao, My Lien, "Collagenase assay" (2019). USF Patents. 1028.
University of South Florida