Rapid bed-side measurement of neutrophil elastase activity in biological fluids

Patent metadata

Field	Value
Publication number	US-10577639-B2
Application number	US-201013376508-A
Country	US
Kind code	B2
Filing date	Jun 28, 2010
Priority date	Jun 26, 2009
Publication date	Mar 3, 2020
Grant date	Mar 3, 2020

Abstract

The subject invention provides novel devices and methods for the detection and quantification of neutrophil elastase activity in biological samples.

First claim

We claim:

1. A method for detection of neutrophil elastase in a fluid from a chronic wound wherein said method comprises the following steps:
a) providing, in a self-contained unit, a single-peptide substrate comprising GABA-Ala-AlaAla-Ala- (SEQ ID NO: 19), to which is attached a fluorescing dye and a quencher, wherein the fluorescing dye is (5-F AM) and the quencher is DABCYL, and wherein the fluorescing dye and the quencher are in sufficiently close proximity on the substrate such that the quencher quenches the fluorescence of the fluorescing dye, and wherein the fluorescing dye and the quencher are separated by the substrate such that, if the neutrophil elastase cleaves the substrate, the fluorescing dye is separated from the quencher,
b) collecting a sample of chronic wound fluid from a patient,
c) in the self-contained unit, contacting the substrate with the fluid within 30 minutes after collection of said fluid for a period of time sufficient to permit substrate cleavage to occur if there is neutrophil elastase present in the fluid; and
d) detecting any fluorescence, wherein the presence of neutrophil elastase in the fluid is indicated by fluorescence.

2. The method, according to claim 1 , wherein results are obtained in less than 1 0 minutes after the sample is collected.

3. The method, according to claim 1 , wherein said method gives a quantitative indication of the amount of neutrophil elastase present in the fluid.

4. The method, according to claim 1 , wherein the contacting step is carried out in the same container as the fluorescence detection step.

5. The method, according to claim 1 , wherein the fluid is collected in a hollow straw and absorbed onto a swab therein,
a solution containing the substrate is then added to the hollow straw, travels through the hollow straw, and contacts the swab containing the fluid, and
the swab containing the fluid and the substrate is inserted into another chamber in which the fluorescence detection step takes place.

6. The method, according to claim 1 , which utilizes only the self-contained unit and a means for causing the fluorescing dye to fluoresce.

7. The method, according to claim 1 , wherein the method is conducted bedside.

Assignees

• Schultz Gregory S
• Gibson Daniel J
• Univ Florida

Inventors

• Schultz Gregory S
• Gibson Daniel J

Classifications

• C12Q1/37 Primary

  involving peptidase or proteinase · CPC title

• G01N2333/966

  Elastase · CPC title

• G01N33/542

  with steric inhibition or signal modification, e.g. fluorescent quenching · CPC title