Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving antigen-antibody binding – specific binding protein...
Patent
1998-03-18
2000-10-17
Housel, James C.
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving antigen-antibody binding, specific binding protein...
435 4, 435 71, 435975, 435961, 536 111, 436543, G01N 33573, G01N 3353, G01N 33531, C12Q 100, C07H 100
Patent
active
061329827
DESCRIPTION:
BRIEF SUMMARY
The invention relates to methods for locating and measuring the activities of compounds such as enzymes, e.g. glycosylhydrolases (enzymes that catalyse the hydrolysis of glycosidic linkages), transglycosylases (enzymes that catalyse transglycosylation), and lyases (enzymes that catalyse the eliminative cleavage of glycosidic linkages).
Living organisms possess a wide variety of glycosylhydrolase and transglycosylase enzymes, which play numerous diverse roles in the lives of the organisms by acting on carbohydrate substrates including xyloglucan, other hemicelluloses, cellulose, starch, glycogen, chitin and pectic polysaccharides. Laboratory methods for detecting and measuring these enzymes usually employ aqueous solutions of appropriate substrates placed in vessels (such as test tubes), which are monitored for enzyme-catalysed changes e.g. by colorimetric, radiochemical, chromatographic or viscometric methods.
However, such methods tend to be time-consuming and do not readily lend themselves to the screening of numerous samples of the enzymes. In addition, the methods do not readily provide spatial information on the distribution or localisation of the enzymes within a specimen such as a gel electrophoretogram or part of an organism.
In one aspect, the present invention provides a method of detecting the presence of a compound of interest, or the spatial distribution within a sample of a compound of interest, the method comprising the step of impregnating or coating a suitable base material with a marker substance capable of reacting or interacting with the compound of interest, the marker substance being labelled in a detectable manner (for example fluorescently, radioactively or being coloured) and having a substantially different affinity for the base material than the product of the reaction or interaction with the compound of interest, the method further comprising the steps of bringing the impregnated base material into contact with the sample under test to permit the labelled marker substance to react or interact with any target compound present in the sample, removing either unreacted labelled marker substance or labelled reacted product with a suitable solvent, and detecting the presence of labelled marker substance remaining on said base material, or detecting the liberation of labelled reacted product from said base material, to give an indication of the presence or absence of the compound of interest.
The invention exploits the ability of some carbohydrates but not others to bind to the base material in the chosen solvent.
Preferably, the compound of interest is an enzyme, and the marker substance is a substrate for the enzyme. Said substrate for the enzyme may be a protein, a peptide, a polysaccharide or an oligosaccharide. In a particularly preferred method, the substrate is an oligosaccharidyl-1-amino-1-deoxy-alditol. These compounds have been developed by the applicants and are believed to be new in themselves. They are referred to where appropriate in this specification as"OADs", which term is to be taken to include O-glycosyl-1-amino-1-deoxy-alditols, O-oligoglycosyl-1-amino-1-deoxy-alditols, O-glycosyl-6-amino-6-deoxy-aldonic acids, O-oligoglycosyl-6-amino-6-deoxy-aldonic acids and/or their lactones, and related products of reductive amination.
It may in some cases be an advantage that the reducing carbohydrate not only possesses one reducing site but possesses two or more reducing sites randomly placed in said reducing carbohydrate. These additional reducing sites may be obtained with an oxidation agent such as sodium periodate.
The invention also provides a method of manufacturing OADs involving the reaction of a reducing carbohydrate with an ammonium salt and a reducing agent. Preferably, the ammonium salt is ammonium hydrogencarbonate and the reducing agent is sodium cyanoborohydride. Further preferred features of the invention are described in the subsidiary claims.
As stated above the method of the invention may be used for detecting enzyme activity and/or for detecting the spatial
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Devi S.
Housel James C.
Novo Nordisk A S
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