Detection of proteases using an immunochemical method with haptenylated-gelatin as a solid-phase substrate.
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 microunits mL(-1) for chymotrypsin, 0-12 microunits mL(-1) for type IX, 0-35 microunits mL(-1) for type XIV and 0-100 microunits mL(-1) for type XXIV. The detection limits of the proteases studied were 89 microunits mL(-1) for chymotrypsin, 0.26 microunits mL(-1) for type IX, 5.8 microunits mL(-1) for type XIV and 6.5 microunits 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.
Abuknesha
Ramadan A
RA
Analytical Sciences Research Group, Pharmaceutical Science Research Division, School of Biomedical & Health Sciences, King's College London, University of London, 150 Stamford St., London SE1 9NH, UK. Ram.abuknesha@kcl.ac.uk
Jeganathan
Fiona
F
DeGroot
Rens
R
Wildeboer
Dirk
D
Price
Robert G
RG
D001426:Bacterial Proteins / Q000032:analysis* / Q000276:immunology; D004797:Enzyme-Linked Immunosorbent Assay / Q000379:methods*; D005780:Gelatin / Q000737:chemistry*; D006241:Haptens / Q000737:chemistry* / Q000276:immunology; D010447:Peptide Hydrolases / Q000032:analysis* / Q000276:immunology; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity; D052616:Solid Phase Extraction / Q000379:methods*
D001426:Bacterial Proteins; D006241:Haptens; D005780:Gelatin; D010447:Peptide Hydrolases