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

Article


Abuknesha, R., Jeganathan, F., DeGroot, R., Wildeboer, D. and Price, R. 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. https://doi.org/10.1007/s00216-010-3540-z
TypeArticle
TitleDetection of proteases using an immunochemical method with haptenylated–gelatin as a solid-phase substrate
AuthorsAbuknesha, R., Jeganathan, F., DeGroot, R., Wildeboer, D. and Price, R.
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.

Research GroupBiophysics and Bioengineering group
LanguageEnglish
PublisherSpringer
JournalAnalytical and Bioanalytical Chemistry
ISSN1618-2642
Electronic1618-2650
Publication dates
Online02 Mar 2010
Print30 Apr 2010
Publication process dates
Deposited25 Mar 2010
Accepted03 Feb 2010
Output statusPublished
Digital Object Identifier (DOI)https://doi.org/10.1007/s00216-010-3540-z
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