Field of the Invention

This invention concerns treatment of carbohydrates, for analytical and other purposes.

Background to the Invention

International Publication No WO88/10422 discloses, inter alia, techniques for analysing carbohydrate structures or distinguishing or separating carbohydrate substances, involving applying carbohydrate substances to an electrophoretic gel and running the gel to cause differential migration of different substances. The carbohydrate substances may be labelled, eσ with a fluorescent labelling reagent, to impart a charge to the substance, thereby to enable electrophoretic separation, and to enable visualisation of the substances after running of the gel. Visualisation may be effected with the naked eye, but enhanced sensitivity is obtained by viewing with a charge coupled device (CCD).

The present invention concerns a development of such techniques in the form of a further processing step applicable to electrophoretically separated carbohydrate units.

Summary of the Invention

According to one aspect of the present invention there is provided a method of treating carbohydrate substances, comprising transferring onto a porous membrane by a blotting technique charged carbohydrate substances which have been run on an electrophoretic gel.

The method enables recovery of charged carbohydrate substances from a gel in a form that is well suited to further treatment or storage, providing a permanent record of electrophoretic separation in the gel. For example, blotted carbohydrate substances can be analysed by probing the blot on the membrane with labelled probes, for instance lectins, sugar specific monoclonal antibodies, viral receptors or other protein probes. Suitable probing techniques are known to those skilled in the art.

The transfer step can be effected using blotting techniques such as are known for blotting proteins from electrophoretic gels, eg as discussed in the paper by Jackson and Thompson in Electrophoresis, 1984, 5_, 35-42. It is preferred to use an electrotransfer or electro- blotting technique, eg using semi-dry blot transfer apparatus, which involves contacting the gel with a suitable membrane and applying an electric field to cause resolved materials in the gel to be transferred to the membrane.

A wide range of porous membranes suitable for use in such techniques are commercially available, and are made of materials including activated paper, nitrocellulose, nylon, and polyvinyl diflurone (PVDF), and charged derivatives of such materials. A suitable membrane for use in a particular system can be selected by experiment.

In a further aspect of the invention provides a method of treating carbohydrate substances, comprising applying charged carbohydrate substances to an electrophoretic gel; running the gel to cause differential migration of different substances; and transferring the substances from the gel onto a porous membrane by a blotting technique.

The gel preferably comprises a relatively dense polyacryla ide gel, having a concentration in the range 15% to 60%, preferably 20% to 40%, although in some cases it may be possible or preferable to use gels of lower concentration.

The gel may be either of uniform concentration or in the form of a gradient gel.

The gel is preferably cross linked, eg with N,N' methylenebisacryla ide (bis).

The presently preferred gel comprises a linear polyacrylamide gradient gel having a polyacrylamide concentration (w/v) varyinα in a continuous αradient from 20% (top) to 40% (bottom). The gel is crosslinked with bis, at a concentration (w/v) varyinα from 0.53% at the lowest concentration of polyacyla ide to 1.06% at the highest concentration of polyacrylamide. Alternatively, the electrophoretic system described in Neville, Jr., D.M., J. Biol. Chem. 1971, 246, 6328-6334 has been found to give good results..

For good sensitivity the gel is preferably run using a stacking buffer system (also known as moving boundary electrophoresis, multiphasic zone electrophoresis and other names), using techniques known for working with

protein and DNA fragments, eg as described in the book

"Gel electrophoresis of proteins: a practical approach" edited by B D Hames and D Rickwood, published by IRL Press.

The carbohydrate substances are preferably labelled with a charged fluorescent labelling reagent prior to electrophoretic separation, thereby to impart a charge to the substance so as to enable electrophoretic separation, and also to facilitate visualisation of the substances after running of the gel and after transfer to the membrane.

The currently preferred fluorescent labels are aminonaphthalenesulphonic acids with one, two or three substituent suphonic acid groups, particularly 8- aminonaphthalene-1,3,6-trisulphionic acid (known as ANTS for brevity). As described in UK Patent Applications Nos. 8921817.6 and 9014158.1 and in a copending PCT application filed on even date under reference C139.1/G (the disclosure of which applications are incroporated herein by reference), use of such labelling materials can enable very good electrophoretic resolution of different carbohydrate substances with very good sensitivity.

The labelling reagent may be attached to sites on the carbohydrate substances, after release if necessary from an attached bio olecule. Alternatively, the biomolecule may be modified in known way to enable incorporation of the labelling reagent.

A carbohydrate substance may be labelled with a labelling reagent such as ANTS by incubating the substance with ANTS and a reducing agent, eg sodium cyanoborohydride. For

good labelling it is found useful to add the ANTS in solution in a mixture of acetic acid and water, eg containing 15 parts by volume of acetic acid to 85 parts by volume of water. ANTS is commercially available, eg from Molecular Probes, Eugene, Oregon, which supplies the material in the form of the disodium salt.

After running the gel the labelled carbohydrate substances when illuminated with ultra violet light of suitable wavelength, may be visible with the naked eye in some cases, although better resolution and sensitivity may be obtained by imaging with CCD. Use of a CCD also has the advantage of giving readily quantitated results very quickly. Further, a CCD can be used to view the gel while it is being run."

It is preferred to use a cooled 2-D CCD, operating in slow scan readout. One example of a suitable CCD system is the CCD 2200 Imaging System produced by Astromed Limited, Cambridge, United Kingdom. The CCD is preferably cooled to at least as low as -25°C, with sensitivity being significantly increased by further cooling down as far as -160°C. Typical operation temperatures are in the range -40°C to -120-C.

The invention is applicable to use on a wide range of carbohydrate structures, including those derived from glycoproteins, proteogylcans, glycolipids, glycosphingolipids, polysaccharides, glycosaminoglycans and other bio olecules, including complex biomolecules containing any of these as a component.

When using ANTS as the labelling reagent, good results have been obtained using an Immobilon-N membrane obtained from Millipore, which is a positively charged derivative of polyvinyl diflurone (PVDF). This material interacts

well with the negatively charged ANTS, and results in substantially 100% retention of ANTS labelled carbohydrate substances on the membrane.

The invention will be further described, by way of illustration, in the following Example and by reference to the accompanying drawings, in which:

Figure 1 is an equation illustrating the reaction of ANTS (shown in ionized condition) with a reducing sugar;

Figure 2 is a photograph of an electrophoretic gel showing various sugars labelled with ANTS;

Figures 3 and 4 illustrate the structures of two complex, branched sugars used in Example 1;

Figure 5 is a photograph of a sheet of Immobilon-N after electrotransfer of ANTS-labelled sugars from the gel of Figure 2;

Figure 6 is a photograph similar to Figure 2, of the gel after blotting; and

Ficure 7 is a photograph similar to Figure 5 of a second sheet of Immobilon-N.

Description of Preferred Embodiments

In the following Example 1, reducing sugars which have been labelled with ANTS are separated by polyacrylamide gel electrophoresis and then transferred (blotted) by electrophoresis perpendicular to the gel onto a thin porous membrane to which they adhere.

The procedure involves the following steps.

Reaction of ANTS with Reducing Sugars

Figure 1 illustrates the reaction of ANTS with a reducing sugar.

The reaction may be achieved by the following procedure.

1. Freeze-dry the reducing sugar(s) in microcentrifuge tube.

2. Add 5ul 0.2M ANTS (in the form of the disodium salt, obtained from Molecular Probes, Eugene, Oregon) in solution in acetic acid/water. (15:85, v/v).

3. Add 5ul 1.0M sodium cyanoborohydride in solution in dimethyl sulphoxide.

4. Mix well and incubate at 37°C for 15 hours (overnight) .

5. Freeze-dry the sample by vacuum.

6. Dissolve the sample in a suitable volume of sample buffer ie 6M-urea, 0.062M-tris-HCI, pK6.8.

7. Either store the sample frozen at -70°C or analyse by electrophoresis immediately.

Electrophoresis of the ANTS Labelled Reducing Sugars

Principle

The ANTS labelled sugars are subjected to electrophoresis in a polyacrylamide gel. The separation is dependent on the molecular weight and the structure of each sugar. A moving boundary (stacking) buffer system is used to give sharp bands and high resloution. This system is based on the method of Laemmli (Nature, 1970, 227, 680-685) but the detergent SDS (sodium dodecyl sulphate) is omitted throughout.

Procedure

1. Set up a vertical slab electrophoretic gel apparatus according to the manufacturers instructions. Use a gel thickness of 0.5mm.

2. Make a resolving gel consisting of a linear polyacrylamide gradient from 20% w/v (top) to 40% w/v. Make the low concentration acrylamide slution 20% w/v and 0.53% w/v N,N' methylenebis acrylamide (bis) and the hiαn concentration solution 40% w/v and 1.06% w/v bis. Both solutions contain 0.38M Tris-HCI buffer pH 8.55, 0.0167% w/v ammonium persulphate and 0.085% v/v N,N,N',N'- tetramethylenediamine (TEMED) . The gel length is 12cm.

3. Overlayer the gel solution with water and allow the resolving gel to polymerise.

4. Make a stacking gel 2cm high on top of the resolving gel. The stacking gel solution consists of 3.0% w/v acrylamide, 0.08% w/v bis, 0.12K Tris-HCI buffer pH6.8, 0-1% w/v ammonium persulphate, 0.1% v/v TEMED. Use a comb to form sample wells in the stacking gel.

5. When the stacking gel is polymerised remove the comb and rinse the sample wells with reservoir buffer (192mM glycine, 25mM Tris base, pH 8.3).

6. Load the samples into the wells.

7. Set up the electrophoretic apparatus according to the manufacturer's instructions.

8. Connect the equipment to a DC power supply and electrophorese.

9. Turn off the current.

10. Remove the gel from the mould and photograph using ultra violet illumination, eg using a light box. The preferred wavelength of illuminating light is about 370nm, but good results have been obtained with a broad spectral spread having a maximum wavelength of 302nm.

This method enables glucose and all of its alphal-4 linked oligo ers from maltose to maltoheptaose to be well separated. In addition it also enables the separation of some onosacharides of the same molecular weight but of different structures eg Glc from Gal and 6-deoxyGlc from 2-deoxyGlc. It will separate maltose from celiobiose and maltotriose from cellotriose but using the present method it has not been possible to separate Glcalphal-3Glc from Glcbetal-3Glc nor Glcalphal-6Glc from Glcbetal-6Glc.

Up to 27 bands have been observed in an enzymic starch digest .(alpha-amylase) on a single gel each differing by a single hexose unit from the next.

Blotting of ANTS Labelled Reducing Sugars

Electrotransfer of ANTS labelled sugars from the gel electrophoretogram to a porous membrane of Immobilon-N is carried out using semi-dry blot transfer apparatus in the following manner.

1. Place a sheet of Whatmann 3MM filter paper wetted with blotting buffer (25mM Tris base 192mM glycine) on the lower electrode of the semi-dry blotting apparatus.

2. Place one or more sheets of blotting membrane for instant Immobilon-N or Immobilon-P (Millipore) on top of the filter paper.

3. Place the gel on top of the membrane.

4. Place 5 sheets of wetted filter paper on top of the gel and press with a printers roller to remove air bubbles.

5. Place the upper electrode of the seir.i-dry blotting apparatus on top of the sandwich.

6. Connect to a DC power pack and electrophorese for 10 minutes at 5V.

There should be no air bubbles between the various layers of the blotting sandwich.

Example 1

Various sugars were labelled with ANTS, as described above, and run on a polyacrylamide electrophoretic gel, as

described above. The resulting gel was photographed under UV illumination on a light box having a broad spectral spread of light with a miximum wavelength of 302nm and the result is shown in Figure 2.

In Figure 2 the tracks are as follows:

1. Standard mixture (see below)

2. Standard mixture as for track 1

3. 6-deoxy-D-Glc, D-Glc, D-Gal, 6-deoxy-L-Gal (L-fucose) maltose, maltoriose, maltoheptaose

4.-14. Starch digested with alpha-amylase (from Bacillus subtilis, obtained from Boehringer).

15. Complex sugar 1 (the structure of which is shown in Figure 3) incubated with beta-galactosidase.

16. Beta-galactosidase.

17. Complex sugar 2 (the structure of which is shown in Figure 4) incubated with beta-galactosidase.

18. Beta-galactosidase

19. Galbetal-4Gal, Galbetal-6Gal, cellotriose

20. Galbetal-4Gal, Galbetal-6Gal, cellotriose 6deoxyGlc, maltoheptaose blotting.

21. As track 3

22. Standard mixture as for track 1.

23. Standard mixture as for track 1.

The standard mixture consists of equal molar quantities of: maltoheptaose, maltohexaose, maltopentaose, maltotetraose, maltotriose, cellotriose, Galbetal-6Gal, maltose, Galbetal-4Glc (lactose), Galalphal-4Gal, GalNac, Gal, Glc,6-deoxyGlc.

The gel of Figure 2 was blotted onto a sheet of Immobilon- N. The membrane after blotting was photographed under UV illumination as before and the result is shown in Figure 5.

Figure 6 is a photograph similar to Figure 2, of the gel after the blotting procedure, showing a small quantity of remaining fluorescent ANTS label.

Figure 7 is a photograph similar to Figure 5 of a second sheet of Immobilon-N membrane placed below (away from the gel) the sheet shown in Figure 5. This second sheet shows no fluorescence owing to the ANTS, indicating that all the labelled sugar molecules have been trapped by the first sheet of immobilon-N.

All of the sugars mentioned in the specification are D isomers unless otherwise specified.