Detection of proteases using an immunochemical method with haptenylated–gelatin as a solid-phase substrate.

Abuknesha, Ramadan A. and Jeganathan, Fiona and DeGroot, Rens and Wildeboer, Dirk and Price, Robert G. (2010) Detection of proteases using an immunochemical method with haptenylated–gelatin as a solid-phase substrate. Analytical and Bioanalytical Chemistry, 396 (7). pp. 2547-2558. ISSN 1360-8592

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Official URL: http://www.springerlink.com/content/7577m55n658168...

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Abstract

A simplified method for the measurement of proteases utilising solid-phase substrates incorporating an ELISA end-point detection step is described. Gelatin–hapten conjugates adsorbed onto polystyrene surfaces were found to be efficient substrates for proteases. Digestion of the solid-phase protein–hapten complexes resulted in proportional desorption of the attached conjugates and decrease in the detectable hapten species. Gelatin–cholic acid conjugates, affinity-purified sheep anti-cholic acid antibody–HRP and a chromogenic substrate were incorporated into a convenient and highly sensitive solid-phase immunochemical method. The detectable signal is inversely proportional to enzyme activity. Bacterial proteases (alpha-chymotrypsin Type II, Type IX from Bacillus polymyxa, Type XIV from Streptomyces griseus, Type XXIV from Bacillus licheniformens) were assayed. Dose–response curves for enzyme activities were measured within ranges of 0–550 µunits mL−1 for chymotrypsin, 0–12 µunits mL−1 for type IX, 0–35 µunits mL−1 for type XIV and 0–100 µunits mL−1 for type XXIV. The detection limits of the proteases studied were 89 µunits mL−1 for chymotrypsin, 0.26 µunits mL−1 for type IX, 5.8 µunits mL−1 for type XIV and 6.5 µunits mL−1 for type XXIV. It was demonstrated that the two-step immunochemical method combines the simplicity and sensitivity of solid-phase enzyme immunoassays, the broad specificity of gelatin as a protease substrate and the flexibility of the solid-phase format.

Item Type:Article
Research Areas:Middlesex University Schools and Centres > School of Science and Technology > Natural Sciences
Middlesex University Schools and Centres > School of Science and Technology > Natural Sciences > Biophysics and Bioengineering group
Citations on ISI Web of Science:1
ID Code:4606
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Deposited On:25 Mar 2010 14:30
Last Modified:04 Dec 2014 13:42

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