title: Optimisation of the detection of bacterial proteases using adsorbed immunoglobulins as universal substrates.
abstract: Bacterial proteases, Type XXIV from Bacillus licheniformens and Type XIV from Streptomyces griseus, were used to investigate the utility and optimisation of a solid phase assay for proteases, using immunoglobulin proteins as substrates. Immunoglobulins IgA and IgG were adsorbed on to surfaces of ELISA plates and exposed to various levels of the bacterial proteases which led to digestion and desorption of proportional amounts of the immunoglobulins. The assay signal was developed by measuring the remaining proteins on the polystyrene surface with appropriate enzyme-labelled anti-immunoglobulin reagents. The assay was fully optimised in terms of substrate levels employing ELISA techniques to titrate levels of adsorbed substrates and protease analytes. The critical factor which influences assay sensitivity was found to be the substrate concentration, the levels of adsorbed immunoglobulins. The estimated detection limits for protease XXIV and XIV were 10micro units/test and 9micro units/test using IgA as a substrate. EC(50) values were calculated as 213 and 48micro units/test for each protease respectively. Using IgG as a substrate, the estimated detection limits were 104micro units/test for protease XXIV and 9micro units/test for protease XIV. EC(50) values were calculated at 529micro units/test and 28micro units/test for protease XXIV and XIV respectively. The solid phase protease assay required no modification of the substrates and the adsorption step is merely simple addition of immunoglobulins to ELISA plates. Adsorption of the immunoglobulins to polystyrene enabled straightforward separation of reaction mixtures prior to development of assay signal. The assay exploits the advantages of the technical facilities of ELISA technology and commercially available reagents enabling the detection and measurement of a wide range of proteases. However, the key issue was found to be that in order to achieve the potential performance of the simple assay, optimisation of the method was essential.
authors.affiliation: Analytical Sciences Research Group, Pharmaceutical Science Research Division, School of Biomedical & Health Sciences, King's College London, London University, London, UK. Ram.abuknesha@kcl.ac.uk
authors.forename: Ram A
authors.initials: RA
authors.lastname: Abuknesha
authors.forename: Fiona
authors.initials: F
authors.lastname: Jeganathan
authors.forename: Dirk
authors.initials: D
authors.lastname: Wildeboer
authors.forename: Robert G
authors.initials: RG
authors.lastname: Price
mesh_terms: D000327:Adsorption; D000818:Animals; D001407:Bacillus / Q000201:enzymology; D004305:Dose-Response Relationship, Drug; D004797:Enzyme-Linked Immunosorbent Assay / Q000379:methods; D006801:Humans; D007070:Immunoglobulin A / Q000737:chemistry; D007074:Immunoglobulin G / Q000737:chemistry; D007136:Immunoglobulins / Q000737:chemistry*; D010447:Peptide Hydrolases / Q000737:chemistry*; D011137:Polystyrenes / Q000737:chemistry; D012756:Sheep; D013305:Streptomyces griseus / Q000201:enzymology; D013499:Surface Properties
chemical_list: D007070:Immunoglobulin A; D007074:Immunoglobulin G; D007136:Immunoglobulins; D011137:Polystyrenes; D010447:Peptide Hydrolases