Blue collagenase assay

Abstract

A method of measuring soluble or insoluble cell or tissue-associated collagenase activity. The substrate includes native (fibrillar) collagen fragments 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.

What is claimed is: 
     
         1 . An assay for measuring soluble or insoluble cell or tissue-associated collagenase activity, comprising:
 obtaining dry collagen fibrils suitable for dying;   staining said collagen fibrils with blue dye at saturation level;   suspending said blue collagen fibrils in a collagenase substrate buffer;   incubating said blue collagen fibril suspension with a test sample at 37° C. on a rotator, wherein collagenase activity results in production of blue collagen particulates that can be readily observed in a resulting mixture;   filtering said mixture to retain said blue collagen fibrils, wherein said hlu collagen particulates are digested and collected in a resulting filtrate;   extracting said blue dye from said filtrate to determine an amount of said digested blue collagen particulates; and   measuring absorbance of said digested blue collagen particulates.   
     
     
         2 . An assay as in  claim 1 , wherein the step of measuring absorbance is performed using a spectrophotometer at an optimal wave length of 600 nm. 
     
     
         3 . An assay as in  claim 1 , wherein the step of staining said collagen fibrils is performed using 0.2% Coomassie Brilliant Blue in a solution of acetic acid-methanol-water (10:40:50, v:v:v), at a ratio of 500 mg collagen in 30 mL acetic acid-methanol-water solution.