Chemistry: electrical and wave energy – Processes and products – Electrophoresis or electro-osmosis processes and electrolyte...
Patent
1996-04-08
1997-06-24
Gorgos, Kathryn L.
Chemistry: electrical and wave energy
Processes and products
Electrophoresis or electro-osmosis processes and electrolyte...
536127, G01N 2726
Patent
active
056413904
DESCRIPTION:
BRIEF SUMMARY
This is a rational stage application of PCT/GB94/01115 filed May 20, 1994.
FIELD OF THE INVENTION
This invention relates to labelled sugars, that can be used in carbohydrate analysis, and to their preparation.
BACKGROUND OF THE INVENTION
For the purposes of carbohydrate analysis, it is known to label oligosaccharides and other carbohydrates with detectable labels. Radio-active labels are widely used, but many prefer to use non-radio-active materials. Various chemical labels have been proposed, e.g. providing fluorescence. Particular fluorescent labels and their use, involving analysis of labelled sugars by differential migration on electrophoresis, are described in WO-A-9428423.
Labelling is generally performed by reaction of the carbohydrate and the labelling reagent in a homogeneous solution. The resultant labelled carbohydrate is then separated.
Immobilised reaction systems are well known. For example, it has been proposed to complex labelling reagents on CELITE. Covalent carbohydrate-support conjugates are also known.
SUMMARY OF THE INVENTION
According to the present invention, a labelled carbohydrate, e.g. monosaccharide or higher oligomer, is provided in the form of an immobilised complex. This is based on the discovery that there are suitable matrix materials which will bind sugars sufficiently, to allow the labelling reaction to proceed and then removal of substantially all contaminants from the immobilised complex of the labelled carbohydrate. This procedure avoids the additional steps associated with the prior art, i.e. "clean-up" steps that are necessary in order to provide the low background required for high sensitivity analysis, especially for capillary electrophoresis. Further, the approach adopted by the present invention reduces transfer and handling steps which can cause loss of sample.
DESCRIPTION OF THE INVENTION
The present invention takes advantage of the fact that sugars are hydrophilic molecules. They are labelled on the surface of a solid matrix such as glass (which is also hydrophilic), in a substantially hydrophobic solvent system. Excess labelling reagent may then be removed by washing the matrix with a hydrophobic solvent such as butanol, leaving the hydrophilic labelled sugars on the matrix surface. The technique therefore relies upon the labelling reagent having some hydrophobic character (which tends to be true for the majority of fluorescent and UV-absorptive labels).
Suitable matrices bind sugars sufficiently to allow the contaminants, i.e. including the reactants used in derivatisation, to be washed away without losing significant amounts of sugar in the washing process. A suitable solid phase matrix is controlled-pore glass (CPG).
In general terms, a conventional derivatisation reagent can be used. Examples are aminomethylfluorescein and 2-aminobenzoic acid (anthranilic acid); see WO-A-9428423, cited above, which discloses 2-aminobenzamide and analogues as labels, and references therein. It is added to the matrix, and derivatisation is allowed to proceed under appropriate conditions, e.g. reductive amination, given the materials involved. Clearly, reagents and conditions should be chosen so that the desired complex is formed, and the contaminants can be washed from it. Suitable materials for the washing step, such as butanol, can also be chosen from among those that are known.
Following derivatisation, sugar components remain bound to the solid phase, and then excess reagents and by-products may be washed away. The labelled sugars may then be eluted from the complex, substantially free of contamination. Again, a suitable eluant, such as water, may be readily selected by the skilled man. In consequence, the desired labelled carbohydrate is obtained in a satisfactory manner, and not in solution until so required.
The following Examples illustrate the invention. Examples 1 and 2 are in the nature of protocols.
EXAMPLE 1
1. Sample (consisting of an equimolar mixture of sugars, e.g. monosaccharides), dried into small tube which contains a small quantity of soli
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Goulding Paul
Hawke David Harry
Gorgos Kathryn L.
Noguerola Alex
Oxford Glycosystems Ltd.
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