Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving antigen-antibody binding – specific binding protein...
Patent
1993-11-29
1995-11-21
Spiegel, Carol A.
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving antigen-antibody binding, specific binding protein...
435 14, 435 18, 435176, 435808, 435817, 4352871, 4352872, 4352887, 436 94, 436524, 436531, 436807, 422 8208, 422 82, 422 11, 356352, 204403, C12Q 134, C12M 140, G01N 3353
Patent
active
054686202
DESCRIPTION:
BRIEF SUMMARY
This invention relates to apparatus and methods for the determination of biomolecules in samples of biological origin, in particular for the characterisation of oligosaccharides on glycoproteins and glycohormones.
BACKGROUND OF THE INVENTION
The ubiquitous distribution of glycoconjugates at cell surfaces, extracellular matrices and within particular organelles has focused a great deal of research on the study of glycoconjugates as molecular determinants in cellular function, intracellular processing and intracellular interactions. Oligosaccharides may be covalently linked to the protein through an asparagine side chain (N-linked) or through a serine or threonine side chain (O-linked).
Differential glycosylation of a polypeptide can create different subsets of glycoproteins which may have different physical and biochemical properties [Rademacher, T. W., Parekh, R. B., and Dwek, R. A. (1988) Annual Review Biochemistry 57, 785-838]. This may result in functional diversity leading to different disease states. For example, there is a shift in the population of the glycosylated forms of IgG towards those with a higher content of agalactosyl biantennary N-linked oligosaccharides in active rheumatoid arthritis, tuberculosis and Crohn's disease. This shift is thought to be involved in disease pathogenesis [Parekh, R. B., Dwek, R. A., Sutton, B. J. et al (1985) Nature 316, 452-457].
One known method of analysis of glycoproteins involves the following steps: oligosaccharide moieties or digestion with a glycopeptidase,
The fractionation profile is compared to the elution parameters of standard oligosaccharide moieties to give a compositional analysis. The oligosaccharide moieties may also be released from the glycoprotein using enzymes such as endoglycosidase H.
An alternative strategy based on fast atom bombardment mass spectroscopy for analysis of glycoproteins has also been developed [Dell, A., Advances in Carbohydrate Chemistry and Biochemistry 45, 19-72].
These methods are time-consuming, and involve the use of radioactivity and/or sophisticated equipment.
We have now devised methods and apparatus for glycosylation analysis which overcome or substantially mitigate certain of the above-mentioned disadvantages, and are particularly useful when very detailed analysis of oligosaccharide composition is not required.
SUMMARY OF THE INVENTION
According to the invention there is provided a method of determining one or more specific oligosaccharide(s) (the analyte) in a sample, which method comprises contacting the sample with a surface on which is immobilised a glycosidase specific for the analyte.
In a first variant of the method according to the invention, there is added, simultaneously with the sample, a substrate for the glycosidase. The substrate is labelled in such a way that it is converted by the glycosidase to a detectable product. The labelled substrate competes with the analyte for binding to the glycosidase, such that the concentration of labelled product is inversely proportional to the concentration of analyte present in the sample.
The types of labels which may be used include substrates that result in a fluorescent, absorbent or luminescent product. For example, many glycosides are available as 4-nitrophenyl-glycosides. The reaction of these with their specific glycosidases results in the release of 4-nitrophenol, a yellow product which absorbs at 405 nm. Many substrates may also be labelled with the fluorogenic group methylumbelliferone which is non-fluorogenic when incorporated into the substrate and fluorogenic when the group is released on reaction with an enzyme. Alternatively, reaction of the substrate with the enzyme may result in the formation of an insoluble product, thereby increasing the refractive index in the vicinity of the surface. This may be detected when the surface is the surface of an optical biosensor. It may also be possible to detect the enzyme reaction directly by electrochemical means.
Several glycosidases specific for different saccharide moieties may be immobilised on the
REFERENCES:
patent: 4857273 (1989-08-01), Stewart
patent: 4865995 (1989-09-01), Dairaku
patent: 5081037 (1992-01-01), Kariyone et al.
patent: 5183743 (1993-02-01), Corey
patent: 5192415 (1993-03-01), Yoshioka et al.
patent: 5192666 (1993-03-01), Ikenaka et al.
patent: 5229833 (1993-07-01), Stewart
patent: 5246846 (1993-09-01), Pittner et al.
Chemical Abstracts, vol. 79, No. 7, 1973, p. 119 Abstract #39596.
Chemical Abstracts, vol. 113, No. 19, 1990. Abstract #168390.
Molloy James O.
Pollard-Knight Denise V.
Fisons plc
Spiegel Carol A.
LandOfFree
Methods and device for glycosylation analysis does not yet have a rating. At this time, there are no reviews or comments for this patent.
If you have personal experience with Methods and device for glycosylation analysis, we encourage you to share that experience with our LandOfFree.com community. Your opinion is very important and Methods and device for glycosylation analysis will most certainly appreciate the feedback.
Profile ID: LFUS-PAI-O-1136196