Classifications

• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K31/00—Medicinal preparations containing organic active ingredients
  • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
  • C07H3/00—Compounds containing only hydrogen atoms and saccharide radicals having only carbon, hydrogen, and oxygen atoms
  • C07H3/06—Oligosaccharides, i.e. having three to five saccharide radicals attached to each other by glycosidic linkages
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K16/00—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies
  • C07K16/18—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies against material from animals or humans
  • C07K16/34—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies against material from animals or humans against blood group antigens
• • G—PHYSICS
  • G01—MEASURING; TESTING
  • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
  • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
  • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
  • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
  • G01N33/80—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving blood groups or blood types or red blood cells

Definitions

• the present invention relates to a process of isolating from different types of cell tissue carbohydrates mainly consisting of oligosaccharides with degradation of glycoconjugates present in the cell tissue, said glycoconjugate being a common term for glycoproteins, proteoglycanes and glycolipids.
• Cell tissue is a complex material consisting inter alia of so-called glycoconjugates, which are constituted by complex structures composed of carbohydrates, proteins and lipids.
• the three main types of glycoconjugates are glycoproteins, proteoglycanates and glycolipids.
• the carbohydrate chains are mostly N-glycosidically bound to asparagine or O-glycosidically to serine, treonine, hydroxylysine or hydr ⁇ xyproline.
• the carbohydrate chains of the proteoglycanes have similar bonds to the protein part.
• the carbohydrate part is O-glycosidically bound and is present as glycosyl ceramides in man and animals.
• the carbohydrate part of cell membrane glycoconjugates has been found to play an essential role in several biological functions including cell-cell interactions, as receptors for antibodies, enzymes, hormones, viruses and toxines.
• cell-cell interactions As receptors for antibodies, enzymes, hormones, viruses and toxines.
• the present invention has for its purpose to provide a process for releasing the carbohydrate part from the glycoconjugates in cell tissue while maintaining the carbohydrate chains in a more or less intact state. More in particular, the invention provides for a process for isolating the carbohydrate fraction from glycolipids of cell tissue by specifically cleaving the glycosidic bond between the carbohydrate part and the ceramide residue.
• the present invention is based on technique residing in treating the cell tissue with a reactant containing or consisting of trifluoroacetic acid anhydride.
• a reactant containing or consisting of trifluoroacetic acid anhydride By this treatment, which can be termed trifluoroacetolysis, the oligosaccharide chains are released from both glycoproteins and glycolipids with simultaneous degradation of proteins and lipids, whereby the oligosaccharides in the form of their trifluoroacetates can be separated and recovered.
• trifluoroacetic acid anhydride solely or trifluoroacetic acid anhydride in combination with trifluoroacetic acid, the latter constituting up to about 90 % by volume of the reactant mixture.
• a preferred reactant is one wherein trifluoroacetic acid anhydride and trifluoroacetic acid are present in about equal proportions based on volume.
• the treatment with the reactant is preferably carried out at an increased temperature and at an increased pressure.
• a preferred temperature range is 80-110°C, for example about 100°C.
• the overpressure in the latter case is about 4 atms.
• the cell tissue is constituted by erytrocyte membranes from which can be recovered oligosaccharides possessing blood group specificity.
• Such blood group specific oligosaccharides recovered in accordance with the technique of this invention can be coupled to carriers with antigenicity, and conjugates obtained thereby may then be used for immunisation for the purpose of obtaining specific antibodies.
• An alternative use of isolated oligosaccharides is coupling of said saccharides to carriers for the preparation of immunosorbent columns which can be used. for purification and isolation of specific antibodies.
• the specific antibodies made can be used in reactants for blood group determination and for other medicinal purposes.
• the reaction mixture obtained when treating the cell tissue with the reactant is suitably evaporized to dryness, and the residue vaporization is distributed between water and a water-immiscible solvent, the aqueous phase containing the carbohydrate fraction being recovered and the solvent phase containing inter alia protein residues and lipid residues being discarded.
• a water-immiscible solvent there may be used for example ether or methylene chloride.
• the erytrocyte membranes are suitably homogenized and suspended in water, whereafter lyophilisation is carried out before the treatment with the reactant.
• the oligosaccharides recovered by the process according to this invention may suitably be purified by gelchromatography and preparative paper chromatography. In this manner there may be obtained from blood of the blood group A the oligosaccharide: ⁇ -GalNAcp-(1-3)-Gal (A-tri) ⁇ -Galp-(1-4)- ⁇ -Galp-(1-4)-Glc or ⁇ -GalNAcp-(1-3)- ⁇ -Galp-(1-4)- ⁇ -Galp-(1-4)-Glc.
• oligosaccharides mentioned under A- and B-blood are obtained from AB-blood.
• the technique according to this invention namely release and recovery of oligosaccharides from erytrocyte membranes which show blood group specificity
• four main steps are part of the procedure, namely the following: a. Isolation of blood group specific oligosaccharides from erytrocyte membranes; b. Chemical modification of isolated oligosaccharide for coupling to an antigenic carrier or a carrier for the preparation of immunosorbent.
• d Purification of antibodies.
• EXAMPLES a Preparation of oligosaccharides. Erytrocytes from discarded blood of blood groups A, B, AB and O are lysed and the membranes are isolated by means of centrifugation. In this way about 10 grams of membranes are obtained from one liter of blood. The membranes are treated according to the process of Dodge, J.T., Mitchell, C, and Hanahan, D.J. (1963),
• reaction mixture is cooled and evaporized to dryness, there being obtained a dark to black-coloured residue.
• residue there is added methanol (700 ml), and the mixture is evaporised to dryness.
• the residue is then diluted with 50 % aqueous solution of acetic acid (1000 ml), and is allowed to star. ⁇ at room temperature for about l8 hours.
• the reaction mixture is filtered with a glass filter and evaporised to dryness.
• the residue obtained is distributed between water and diethyl ether.
• the ether phase is washed 4 times with water, and the combined wash solutions are washed with diethyl ether 4 times.
• the aqueous solution obtained is yellow-coloured and contains the released oligcsaccharides.
• the oligosaccharides obtained are in such form that the N-acetyl functions have been converted to N-trifluoroacetyl groups.
• the oligosaccharides obtained can be purified by gel chromatography followed by a preparative paper chromatography, and the purification procedure is continuously surveyed with gas chromatography mass spectrometry.
• the following oligosaccharides are obtained in the present example by treatment of blood originating from blood groups A, B, AB and O with varying blood groups within the P-system:
• Chemical modification of isolated oligosaccharides In order that the oligosaccharides shall be capable of coupling to carriers of different kinds they should be chemically modified. Such modification can be performed by means of known technique. Before the chemical modification it is suitable to de-N-trifluoroacetylate the oligosaccharides, which is suitably carried out in a basic environment at a pH of about 10-12 and at room temperatures for a period of time of about 20 hours. This results in the formation of amino groups, which can be N-acetylized to reform the N-acetyl functions.
• the aliphatic amino groups formed by the de-N-trifluoroacetylation can be used for coupling the modified oligosaccharide to different carrier materials.
• the modified oligosaccharides can be coupled to proteins, which can be constituted by albumen or edestine, and possibly to lipid A (from lipopolysaccharide) for the purpose of obtaining a high yield of M-antibodies.
• the conjugates obtained are then used for immunising animals, for example rabbit or horse, or for the production of antibodies by hybridom technique (Kohler, G. and Milstein, C, 1975, Nature (London) 256, pp. 495-497; Kohler G., and Milstein, C, 1976, Eur.J. Immunol. 6 pp. 511-519).
• the antisera thereby obtained are then serologically used in connection with known blood classification methods.
• d Purification of antibodies.
• Chemically modified blood group active oligosaccharides prepared according to the above are coupled to a suitable carrier material, for example Sephadex, and then sera from immunised animals are passed through an immunosorbent column formed by the treated Sephadex.
• Antibodies having specificity vis-a-vis coupled oligosaccharide adheres by reaction with same, whereas materials of no interest are washed away.
• the specific antibodies adhering to the column by reaction with coupled oligosaccharides are then desorbed, and antibody solutions of unambiguously defined specificity are obtained.
• Purified specific antibodies ar ⁇ then tested for specificity for control and may then be used for blood group determination by hemagglutination technique.
• the present invention is not delimited to the exemplary application for preparation of blood group active oligosaccharides from erytrocyte membranes as described above.
• the invention is applicable to all types of cell tissue for recovery and isolation of oligosaccharides for different purposes.
• epithelium tissue intestinal mucous membrane, urinary tract epithelium, lung epithelium etc.
• isolated oligosaccharides can be of diagnostic importance.
• GalNAcp 2-acetamido-2-deoxy-D-galactopyranosyl
• Gal D-galactose

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• Urology & Nephrology (AREA)
• Biomedical Technology (AREA)
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• Genetics & Genomics (AREA)
• Proteomics, Peptides & Aminoacids (AREA)
• Food Science & Technology (AREA)
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• Pharmacology & Pharmacy (AREA)
• Animal Behavior & Ethology (AREA)
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• Epidemiology (AREA)
• Veterinary Medicine (AREA)
• Physics & Mathematics (AREA)
• Analytical Chemistry (AREA)
• General Physics & Mathematics (AREA)
• Pathology (AREA)
• Saccharide Compounds (AREA)
• Polysaccharides And Polysaccharide Derivatives (AREA)
• Medicines Containing Material From Animals Or Micro-Organisms (AREA)
• Investigating Or Analysing Biological Materials (AREA)

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• 1980
  • 1980-01-28 SE SE8000658A patent/SE8000658L/xx not_active Application Discontinuation
• 1981
  • 1981-01-28 IT IT19379/81A patent/IT1141962B/it active
  • 1981-01-28 WO PCT/SE1981/000021 patent/WO1981002104A1/en not_active Ceased
  • 1981-01-28 EP EP81900324A patent/EP0051593A1/de not_active Withdrawn
  • 1981-01-28 JP JP56500550A patent/JPS57500022A/ja active Pending
  • 1981-09-25 DK DK427581A patent/DK427581A/da not_active Application Discontinuation

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