Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or...
Patent
1997-05-14
1999-08-03
Kemmerer, Elizabeth
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
435 23, 435 24, 530323, 530331, 530330, 530329, G01N 3348
Patent
active
059324104
DESCRIPTION:
BRIEF SUMMARY
ASSAY FOR THE DETECTION OF PROTEASE
The present invention relates to a an assay for the detection of active proteases in samples, particularly samples of biological origin. In addition, the present invention relates to substrates having protease specificity.
During a study of mutant proteases the need arose for an assay method that could rapidly and accurately determine the activity and specificity of a large number of serine proteases produced in bacterial cultures.
Proteases of the serine (and thiol) families possess esterase activities which proceed at much faster rates than the cleavage of peptide bonds. Advantage was taken of this activity to develop an "esterase" assay which uses a pH indicator to visualise the hydrogen ion release caused by the action of proteases on peptide-ester substrates. A series of twenty compounds of the form, carbobenzoxy alanyl-X-methyl ester (Z-AX-OMe) were synthesised where "X" is one of the naturally occurring amino acids. These substrates were used in a 96 well microtitre plate assay to screen each mutant enzyme for activity and specificity. The reactions were followed visually or quantified using a standard ELISA plate reader. The esterase assay, using trypsin as a test enzyme and Z-Ala-Arg-OMe as substrate, was more sensitive as than traditional trypsin substrates (three times the rate obtained with benzoyl arginine ethyl aster and twenty times the rate of benzoyl arginine p-nitroanilide) (Whittaker et al., Analytical Biochemistry, 220, 238-243, 1994).
The success of this assay led us to question its possible broader use in the characterisation and quantification of proteases in research in general and in clinical enzymology in particular. For example the assay could be useful for the rapid characterisation of a protease/s being produced by tumours where comparison of the reaction profile against a selected species of ester substrates would identify and quantify the proteases and potentially give an indication of the tumour's metastatic potential.
A number of difficulties arise in using such an assay system for biological samples. These include the buffering ions in the sample and that some proteases are present at only very low levels. In order to at least partly ameliorate theme problems and to improve the specificity the present inventors have developed an assay method involving ligand capture of the protease. In one form this is achieved by combining an ELISA/Dot Immunobinding Assay (Allergy 50 (2); 119-125, 1995,Y. Yu) and the Esterase Assay.
Accordingly, in a first aspect the present invention consists in a method of detecting the presence of an active protease in a sample, the method comprising the following steps: protease; protease; peptide ester substrate which is hydrolysed by the protease; and substrate.
The ligand may be any of a number of binding molecules well known in the art. It is however, necessary that the ligand binds to the protease at a position which is not involved in the hydrolysis of the ester. It is presently preferred that the ligand is an antibody, preferably a monoclonal antibody or antibody fragment.
The peptide ester substrate may be any of a number of substrates hydrolyzed by the protease. Preferably, the substrate is hydrolysed rapidly by the protease.
By appropriate selection of the peptide ester substrate it in possible to add a further layer of specificity to the assay method. For example, use of ligand reactive with prostate specific antigen and a substrate which is specifically hydrolysed by prostate specific antigen will result in a highly specific assay method.
In a further preferred embodiment the production of a proton by the hydrolysis of the peptide ester is detected using a pH indicator. A pH indicator, such as phenol red, may be added after step 4, however, it is presently preferred that indicator is included in the solution added in step 4.
In another preferred embodiment the sample is serum or semen.
The method of the present invention could be used to detect the presence of a number of important mammalian proteases.
REFERENCES:
patent: 5459035 (1995-10-01), Guerrero et al.
Whittaker, et al. Anal. Biochem. vol. 220: pp. 238-243, 1994.
Kuriyama, et al. Cancer Research vol. 41(10): pp. 3874-3876, Oct. 1981.
Whittaker Robert G
Yu Yan
Commonwealth Scientific and and Industrial Research Organization
Kemmerer Elizabeth
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