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United States Department of Agriculture

Agricultural Research Service

Title: Colorimetric Detection of Human Leukocyte Elastase with Modified Cellulose Dipsticks

Authors
item Pierre, Sonya
item Edwards, Judson
item Cohen, I. Kelman - MCV WOUND HEALING CTR
item Yager, Dorne - MCV WOUND HEALING CTR

Submitted to: Bioconjugate Chemistry
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: April 13, 2003
Publication Date: N/A

Interpretive Summary: The technology for determining the rate of wound healing in chronic wounds is necessary. This research focuses on developing a spot test in the form of a dipstick to determine the rate of wound healing. Elastase, an enzyme, present in a chronic wound is the target of interest for detection by use of a chromogenic substrate immobilized to cellulose. Since, high levels of elastase in the wound have been associated with destroying tissues that are necessary for wound healing, these levels should be monitored. Therefore, elastase is detected by using a dipstick that is specifically designed to detect the enzyme levels in the wound when chromophores from the immobilized substrate are released. This technology will benefit doctors and nurses in determining the rate of wound healing and when bandages need changing.

Technical Abstract: High levels of human neutrophil elastase (HNE) in chronic wounds have been associated with degradation of cytokine growth factors necessary for normal wound healing. Thus, accurate clinical detection and quantification of HNE will be important to the therapeutic management of chronic wounds. Colorimetric detection of HNE using a rapid spot test to detect HNE levels on an enzyme substrate-derivatized cellulose has been developed. The chromogenic peptide substrate succinyl-Ala-Ala-Pro-Val-pNA and its analog succinyl-Ala-Ala-Pro-Ala-pNA were attached to derivatized cellulose. Cellulose was treated with 3-aminopropyltriethoxysilane to form the amino-propyloxy-ether (AP) of cellulose. The amino-propyloxy-ether of cellulose was reacted with the HNE chromogenic peptide substrate to form a conjugate of cellulose (cel-AP-suc-Ala-Ala-Pro-Val-pNA) through an amide linkage. Amino acid analysis of the peptide-cellulose products revealed from 7.8 - 14.5 mmol/g of substrate per gram of derivatized cellulose. The colorimetric response of the cellulose-bound chromophore was assessed in HNE buffered solutions by monitoring release of para-nitroaniline onto a derivatized paper surface. Both visual and spectral detection methods were used to determine elastase levels from 0.025 to 30.0 µg/mL. A comparison of the analogs demonstrated cel-AP-suc-Ala-Ala-Pro-Val-pNA gave stronger absorption than the cel-AP-suc-Ala-Ala-Pro-Ala-pNA.

Last Modified: 10/25/2014
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