Chemistry: electrical and wave energy – Processes and products – Electrophoresis or electro-osmosis processes and electrolyte...
Reexamination Certificate
1998-04-24
2001-02-20
Marschel, Ardin H. (Department: 1631)
Chemistry: electrical and wave energy
Processes and products
Electrophoresis or electro-osmosis processes and electrolyte...
C204S456000, C204S466000, C436S094000, C436S095000, C536S123000, C536S055200, C534S617000, C435S014000
Reexamination Certificate
active
06190522
ABSTRACT:
1.0 BACKGROUND OF THE INVENTION
1.1 Field of the Invention
The present invention is generally directed to the field of carbohydrate analysis, and more particularly to the analysis of carbohydrates derivatized with a visible chromophoric dye by high resolution polyacrylamide gel electrophoresis. The invention also includes methods and kits for analyzing carbohydrates.
1.2 Description of Related Art
Carbohydrates have many important biological functions. Carbohydrates play important roles in development, immunology and cancer metastasis (Shur, 1994; Wright and Morrison, 1997; Kawaguchi, 1996). Glycoproteins are involved in cell stability and adhesion, antibody recognition, and microorganism binding, and also serve as cell-surface markers (Wyss and Wagner, 1996; Hounsell et al., 1996; Parham, 1996).
Thus, there is substantial interest in carbohydrate analysis. However, a technique for carbohydrate analysis that is both inexpensive and easily performed is currently unavailable. High-sensitivity carbohydrate analysis has been performed with gas chromatography (Karlsson et al., 1995; Karlsson et al., 1994; Hansson and Karisson, 1993), liquid chromatography (Starr et al., 1996; Hu, 1995; Weitzhandler et al., 1993), mass spectrometry (Reinhold et al., 1996; Kovacik et al., 1996; Hellerqvist and Sweetman, 1990), nuclear magnetic resonance spectroscopy (Sugahara et al., 1996; Larnkjaer et al., 1995; Pozsgay and Coxon, 1994; Haynes et al., 1992) and capillary electrophoresis (El Rassi and Mechref, 1996; Oefner and Chiesa, 1994; Liu et al., 1991). However, the above methods of carbohydrate analysis require sophisticated instrumentation and highly trained personnel.
A method for carbohydrate analysis that utilizes polyacrylamide gel electrophoresis to separate fluorescently-labeled carbohydrates has been developed (Jackson, 1996; Jackson, 1994a; Jackson, 1994b) and optimized (Klockow et al., 1996; Masada et al., 1996). However, fluorometers for detection and quantification of the modified carbohydrates are not widely available. Thus, a technique for carbohydrate analysis that is both inexpensive and easily performed is currently unavailable.
2.0 SUMMARY OF THE INVENTION
It is, therefore, a goal of the present invention to provide a simple, inexpensive method for analyzing carbohydrates that does not require the use of a fluorometer. The method of carbohydrate analysis disclosed herein involves the labeling of carbohydrates with a visible dye followed by electrophoretic separation of the dye-labeled carbohydrates. The visible carbohydrate-dye adducts are then identified by their migration or mobilities in the gel and quantified by, for example, densitometric analysis. This method is easy to use and employs commonly available instruments for the separation of the labeled carbohydrates and subsequent analysis.
An embodiment encompasses a method of detecting the presence of a carbohydrate in a sample suspected of containing a carbohydrate by contacting the sample with a chromophore to produce a labeled carbohydrate; separating the labeled carbohydrate from any unreacted chromophore by gel electrophoresis; and detecting the labeled carbohydrate. The identity of any carbohydrates present may then be determined based on its mobility, for example, by a comparison of the migration of the labeled carbohydrate in the gel with known standards, such as known carbohydrates, run under similar conditions. This method allows for the detection and identification of carbohydrates present in the sample in subpicomole quantities.
As used herein “a” will be understood to mean one or more.
The disclosed method typically involves the labeling of carbohydrates, such as mono-, di-, tri-, tetra-, hepta- and even poly-saccharides, with a chromophore. Although the method may be employed to analyze samples containing only one carbohydrate, it may be preferable to employ this method with samples suspected of containing mixtures of two or more carbohydrates.
The chromophore used to label the carbohydrates may be any visible dye capable of derivatizing or labeling the carbohydrate. It should have an absorption maximum in the visible region, which preferably has a high extinction coefficient. Dyes having higher extinction coefficients allow for more sensitive detection. For example, 4-amino-1,1′-azobenzene-3,4′-disulfonic acid (“AABA”) has an absorption maximum at 489 nm, with an extinction coefficient of 37,615 which facilitates the visible detection of labeled carbohydrates at low levels (approximately 20 nmol).
The chromophore may also be charged or coupled to a charge-imparting species. It is envisioned that such “charged” chromophores will be particularly useful for the separation and identification of neutral carbohydrates. The charge-imparting chromophore facilitates the labeled carbohydrate's migration in an electric field and thus, the efficient separation by polyacrylamide gel electrophoresis. However, the use of uncharged chromophores is also envisioned particularly for the separation and identification of charged carbohydrates or with the use of additional charged species that may be associated or linked to the uncharged chromophore or carbohydrate.
Embodiments include the labeling of carbohydrates with chromophores such as 4-amino-1,1′-azobenzene or 4′-N,N-dimethylamino-4-aminoazobenzene and their sulfonic acid derivatives. For example, the chromophore may be 4-amino-1,1′-azobenzene-3,4′-disulfonic acid or 4′-N,N-dimethylamino-4-aminoazobenzene-2′-sulfonic acid and the like. Other chromophores which may be employed with aspects of this invention include aminoazobenzene, Direct Red 16, CI Acid Red 57, CI Acid Blue 45, CI Acid Blue 22, CL Mordant Brown 13, and CI Direct Orange 75.
The labeled carbohydrates are produced by contacting the chromophore with the sample under conditions effective to label the carbohydrate with the chromophore by producing a chromophore-dye adduct The carbohydrates may be labeled by any method that is known to those of skill in the art, such as reductive amination and other methods disclosed in U.S. Pat. No. 5,668,272 to Prasad et al., herein incorporated by reference. For example, if a carbohydrate is labeled by a reductive amination process, the reducing end of the sugars are reductively aminated in an aqueous solution with an appropriate chromophore.
The labeled carbohydrates may then be separated by polyacrylamide gel electrophoresis on the basis of size, structure, charge, and hydrophobicity. This method permits the separation of stereoisomers and enantiomers. For example, the sample may contain mixtures of mono-, di-, tri-, and hepta-saccharides; at least two different monosaccharides; mixtures of monosaccharides with their derivatives, such as glucose and glucose derivatives; and combinations thereof.
After the carbohydrates have been labeled, the sample is subjected to polacrylamide gel electrophoresis, or similar electrophoretic separation means, in order to separate and concentrate the carbohydrate-dye adducts into bands. The electrophoresis should be conducted for a time sufficient to allow for separation of the labeled carbohydrate and resolution into bands. This separation step generally results in the separation of the labeled carbohydrates from any unreacted chromophore.
Electrophoresis may proceed past the point where some carbohydrates have been removed from the electrophoresis separation medium. Thus, it may be preferable to proceed with the separation and detection in stepwise fashion. For example, some embodiments include labeling any carbohydrates present with a chromophore; partially separating the labeled carbohydrates from any unreacted chromophore and each other; and then halting the separation and detecting the labeled carbohydrates. The separation of the labeled carbohydrates may then be resumed for subsequent detection steps. Furthermore, these steps may be repeated in stepwise fashion.
The concentration and length of the polyacrylamide gel to be employed depends upon the suspected identity of the carbohy
Board of Regents , The University of Texas System
Fulbright & Jaworski L.L.P.
Marschel Ardin H.
Moran Marjorie A.
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