Claims for Patent: 10,428,366

Summary for Patent: 10,428,366

Title:	Collagenase assay
Abstract:	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.
Inventor(s):	Dao; My Lien (St. Petersburg, FL)
Assignee:	University of South Florida (Tampa, FL)
Application Number:	15/210,239
Patent Claims:	1. A method of measuring bacterial collagenase activity in a sample, comprising: staining native collagen fibrils with a dye at saturation level, said collagen fibrils being dry or lyophilized, said dye being capable of stably staining said collagen fibrils; suspending said dyed collagen fibrils in a collagenase substrate buffer; incubating said dyed collagen fibril suspension at about 37.degree. C., wherein bacterial collagenase activity results in digestion of said dyed collagen fibrils into smaller, dyed collagen particulates that can be readily observed in a resulting mixture; filtering said mixture to retain said dyed collagen particulates, wherein said dyed collagen particulates are digested and collected in a resulting filtrate; extracting said dye from said filtrate to determine an amount of said digested dyed collagen particulates; measuring absorbance of said extracted dye to determine an amount of said digested collagen particulates; wherein said native fibrils are not solubilized by acid or gelled by heat, and wherein said native fibrils are Type I collagen found in an animal body. 2. A method as in claim 1, further comprising: prior to the step of staining said dried collagen fibrils, drying said native collagen fibrils, cutting said fibrils into shorter fragments, collecting said fibril fragments as dry collagen. 3. A method as in claim 1, wherein said dye is a triphenylmethane dye. 4. A method as in claim 3, wherein said triphenylmethane dye has a structure of formula I ##STR00002## 5. A method as in claim 4, wherein the step of staining said collagen fibrils is performed using said formula I dye in a solution of acetic acid-methanol-water (1:4:5, v:v:v), at a ratio of 500 mg collagen in about 2-3 ml said acetic acid-methanol-water solution. 6. A method as in claim 1, wherein the step of measuring said absorbance of said extracted dye is performed using a plate reader or a spectrophotometer at an optimal wave length of about 600 nm. 7. A method as in claim 1, wherein said sample includes soluble bacterial collagenase, insoluble bacterial cell-associated collagenase, or insoluble tissue-associated bacterial collagenase. 8. A method as in claim 1, wherein said sample includes a bacteria or biological sample. 9. A method as in claim 1, further comprising quantifying said bacterial collagenase activity by a concentration of said extracted dye, where said concentration is proportional to a concentration of said collagen particulates released. 10. A method as in claim 1, wherein said collagen fibrils are insoluble. 11. A method as in claim 1, wherein the step of filtering said mixture is performed through glass wool which retains any undigested collagen fibrils. 12. A method as in claim 1, wherein the step of extracting said dye from said filtrate is performed by adding an organic solvent to said filtrate and centrifuging the resulting mixture. 13. A method as in claim 12, wherein said organic solvent is detergent polysorbate 20 or a formamide. 14. A method as in claim 1, wherein a quantification of said bacterial collagenase activity is used to detect and quantify true collagenase activity in invasive pathogenic bacteria for development of drugs or vaccines to prevent invasive bacterial disease by blocking bacterial collagenase activity. 15. A method as in claim 1, wherein said collagenase substrate buffer comprises Tris, NaCl, and CaCl.sub.2 in an approximate 5:5:1 ratio by concentration at a pH of about 7.5; or a phosphate buffer of NaCl, KCl, Na.sub.2HPO.sub.4, and KH.sub.2PO.sub.4 in deionized water at a pH of about 7.5. 16. A method as in claim 1, wherein said dyed collagen fibrils are further suspended in sodium azide. 17. A method as in claim 1, wherein the step of extracting said dye includes removing excess dye by rinsing with a solution of acetic acid and methanol in deionized water. 18. A method as in claim 1, further including incubating said dyed collagen fibrils with trypsin to ensure specificity. 19. A method of measuring bacterial collagenase activity in a bacterial or biological sample, wherein said sample includes soluble bacterial collagenase, insoluble bacterial cell-associated collagenase, or insoluble tissue-associated bacterial collagenase, the method comprising: lyophilizing or drying native, insoluble collagen fibrils; cutting said fibrils into shorter fragments; collecting said fibril fragments as dried collagen fibrils; staining said dried collagen fibrils with a triphenylmethane dye at saturation level in a solution of acetic acid-methanol-water (1:4:5, v:v:v), at a ratio of 500 mg collagen in about 2-3 mL said acetic acid-methanol-water solution, wherein said native fibrils are not solubilized by acid or gelled by heat, wherein said native fibrils are Type I collagen found in an animal body, said triphenylmethane dye having a structure of formula I ##STR00003## suspending said dyed collagen fibrils in sodium azide and a collagenase substrate buffer including Tris, NaCl, and CaCl.sub.2) in an approximate 5:5:1 ratio by concentration at a pH of about 7.5; incubating said dyed collagen fibril suspension at about 37.degree. C., wherein collagenase activity results in digestion of said dyed collagen fibrils into smaller, dyed collagen particulates that can be readily observed in a resulting mixture; filtering said mixture through glass wool to retain said dyed collagen particulates, wherein said digested dyed collagen particulates are collected in a resulting filtrate, said glass wool retaining any undigested collagen fibrils; extracting said dye from said filtrate to determine an amount of said digested dyed collagen particulates, by adding polysorbate 20 to said filtrate, centrifuging the resulting mixture; and measuring absorbance of said extracted dye in the supernatant to determine an amount of said digested collagen particulates, wherein said measurement is taken using a plate reader or a spectrophotometer at an optimal wave length of about 600 nm; quantifying said collagenase activity by a concentration of said extracted dye, where said concentration is proportional to a concentration of said collagen particulates released, wherein a quantification of said collagenase activity is used to detect and quantify true collagenase activity in invasive pathogenic bacteria for development of drugs or vaccines to prevent invasive bacterial disease by blocking collagenase activity; incubating dyed collagen substrate with trypsin instead of bacterial collagenase to ensure specificity.

Details for Patent 10,428,366

Applicant	Tradename	Biologic Ingredient	Dosage Form	BLA	Approval Date	Patent No.	Expiredate
Smith & Nephew, Inc.	SANTYL	collagenase	Ointment	101995	06/04/1965		2034-07-02