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• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
  • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
  • C12Q1/34—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving hydrolase
  • C12Q1/44—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving hydrolase involving esterase
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
  • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
  • C12Q1/70—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving virus or bacteriophage
• • 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
  • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
  • G01N33/5308—Immunoassay; Biospecific binding assay; Materials therefor for analytes not provided for elsewhere, e.g. nucleic acids, uric acid, worms, mites
• • 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
  • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
  • G01N33/569—Immunoassay; Biospecific binding assay; Materials therefor for microorganisms, e.g. protozoa, bacteria, viruses
  • G01N33/56983—Viruses
• • 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
  • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
  • G01N33/574—Immunoassay; Biospecific binding assay; Materials therefor for cancer
  • G01N33/57469—Immunoassay; Biospecific binding assay; Materials therefor for cancer involving tumor associated glycolinkage, i.e. TAG

Definitions

• the invention relates to a method for the detection and / or quantitative determination of 9-N / O-acetylated sialic acids, which are bound to carrier molecules.
• the carrier molecules can be carbohydrates, in particular complex carbohydrates, cells, tissues or other biological materials.
• the sialic acids can be synthetic or naturally 9-N / O-acetylated.
• Neuraminic acid is widely used as an N- and O-acetylated compound.
• Sialic acids are found everywhere in the human body, especially in the blood, in slimy secretions (mucins) and in cell membranes.
• mucins slimy secretions
• cell membranes As a component of glycoproteins and glycolipids, they cover all cells of higher animals and humans with an electronegative layer and have a variety of biological functions there. Some of these functions are closely related to certain modifications of N-acetylneuraminic acid, particularly their naturally occurring esterification with acetic acid at position 9 ⁇ 9-O-acetylation).
• 9-O-acetylated ganglioside GD3 is a tumor marker in malignant melanoma in humans. In human breast carcinoma increased formation of Neu ⁇ , 9Ac 2 was found on T lymphocytes. Changes also occur during the development of the brain and other mammalian tissues and cells. 9-O-acetylation is reduced in tumors of the human colon. There are also lectins with a specificity for 9-0-acetylated sialic acids.
• influenza C viruses can bind to 9-O-acetylated sialic acids.
• influenza C viruses also infect human upper respiratory tract and cause mild febrile infections in children up to the age of 10. Because of the high antibody titers in the blood, older people are largely protected against infections with influenza C viruses, so that no expensive protective measures are necessary for working with these viruses.
• the influenza C viruses can be propagated relatively easily in certain cell cultures or in fertilized chicken eggs. The associated working methods or working techniques are well known to the person skilled in the art. These visas are generally accessible viruses.
• the influenza C viruses are surrounded by a membrane envelope in which several copies of a viral glycoprotein are anchored in the form of so-called "spikes".
• Three mutually independent activities are located on this glycoprotein, which play an important role in the infection of the host cells.
• it is hemagglutinin, which represents a section on the molecule with lectin character. Hemagglutinin specifically recognizes 9-N / O-acetylated sialic acids as receptor determinants and mediates the binding of the viruses to their host cells.
• Another activity is sialat 9-0-acetylesterase.
• influenza C viruses have also been made to use influenza C viruses as an "analytical instrument" for the detection or determination of 9-O-acetylated sialic acids.
• the problem arises that the viral esterase cancels the virus binding by splitting off the 9-O-acetyl groups.
• Attempts have therefore already been made to specifically inhibit the esterase activity of the influenza C viruses with the highly toxic esterase inhibitor diisopropyl fluorophosphate (DFP) in order to prevent the binding of biotin-labeled viruses to red blood cells
• DFP diisopropyl fluorophosphate
• the object of the present invention is to provide an improved method for the detection and / or quantitative determination of 9-N / O-acetylated sialic acids bound to carrier molecules.
• the method according to the invention represents a biological test system which directly supports the specific binding of influenza C viruses or coronary viruses to naturally or synthetically 9-N / O-acetylated sialic acids, which can be bound to carrier molecules the viral O-acetylesterase is detected enzymatically.
• Carbohydrates, in particular complex carbohydrates, cells, tissues and any other biological material can serve as carrier molecules. It is also possible to detect or determine naturally occurring free 9-0-acetylated sialic acids and artificially synthesized 9-N-acetylated sialic acids.
• the viruses be used as a unit, but it is also possible to use the viral glycoproteins in isolated or recombinant form.
• the method according to the invention it is possible, for example, to detect the general presence of 9-N / O-acetylated sialic acids. It is also possible to quantitatively estimate the amount of receptors present.
• substance identification with previous separation (for example by electrophoresis, thin-layer chromatography).
• the method according to the invention utilizes two of the biological activities of the viral glycoprotein described above, namely on the one hand the specific binding of the viruses to carrier molecules, in particular complex carbohydrates, with 9-N / O-acetylated sialic acids via their hemagglutinin and on the other hand the Esterase activity of the bound viruses for their detection.
• the influenza C viruses or coronary viruses are bound to the carrier molecules at a temperature at which the esterase is practically inactive. It is preferably carried out at a temperature of 2 to 6 ° C and in particular at about 4'C. One can also work at temperatures Tempera ⁇ , the above 6 * C are, for example up to 10 'C. However, the higher the temperature, the more active the esterase.
• the carrier molecules carrying the sialic acids are preferably also immobilized on a carrier material. This is then washed thoroughly after the viruses have bound, so that all viruses that are not bound or are bound non-specifically are washed away.
• a particularly synthetic esterase substrate is offered, for example 4-methylumbelliferyl acetate (MU-Ac) or ⁇ -naphthyl acetate.
• MU-Ac 4-methylumbelliferyl acetate
• ⁇ -naphthyl acetate a particularly synthetic esterase substrate
• the temperature is raised to such values may be in which proceed the enzymatic reaction, for example at 37 "C or to room temperature. Only where viruses are attached, a fluorescence or a color reaction developed. Since the Viruspar ⁇ Tikel numerous Glykoproteinspikes on have an enhancement effect that cannot be achieved with isolated glycoproteins. The detachment of the viruses from their receptors due to the esterase activity no longer plays a role at this point in time. The virus remains trapped in the individual wells that are no longer washed.
• the chromogenic substrate ⁇ -naphthyl acetate is preferably used, which is cleaved many times more quickly than any natural substrate by the viral esterase.
• the free naphthol immediately reacts with diazonium ions to form an insoluble dye that precipitates where the viruses are bound.
• the methods for detecting and / or quantifying the esterase-substrate reaction are otherwise known.
• Influenza C viruses are preferably used in the method according to the invention, since their affinity for 9-O-acetyl-N-acetylneuraminic acid (eu5.9Ac2) is greater than that of coronary viruses.
• the specificity of the influenza C virus hemagglutinin for 9-N / O-acetylated sialic acid could also be adequately secured with the aid of the method according to the invention.
• saponification of the ester group means that virus binding is completely lost.
• influenza C viruses recognize both gangliosides and glycoproteins as diverse as mucins (mucus), serum glycoproteins or membrane-bound glycoproteins, provided that they have 9-N / 0-acetylated ones Sialic acids.
• a highly specific test for 9-N / O-acetylated sialic acids is thus provided, which can be bound to a variety of carrier molecules.
• the bound virus is directly detected by its enzyme activity.
• the viruses are not labeled by radioactive isotopes, bio-tin or digoxigenin.
• the method according to the invention allows both a qualitative and a quantitative evaluation, quantitative being understood to mean both semi-quantitative and fully quantitative.
• the sensitivity of the method according to the invention is extremely high. For example, with a 9-0-acetylated ganglioside (GDla) as the receptor, immobilized on polystyrene microtiter plates, with MU-Ac as substrate 20 pg (57 fmol) Neu ⁇ , 9Ac2 could be detected.
• GDla 9-0-acetylated ganglioside
• ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇
• test system according to the invention can also be combined with antibodies or lectins.
• process according to the invention is explained in more detail with reference to the following examples which describe preferred embodiments:
• Microtiter plates Immo-Modules Maxisorp F8, Nunc, Roskilde, Denmark
• Fluori eter 1000 M, Perkin-Elmer, Sprint, or a device for fluorescence measurement in microtiter plates.
• Buffer (a) PBS, pH, 2 8.0 g * NaCl
• Influenza C viruses eg strain Johannesbur (1/66) are grown and cleaned as described. The viruses are grown in fertilized chicken eggs at 33 ° C. and the allantoic fluid is removed after 3 days. The viruses are removed by differential centrifugation enriched in two steps, was added in PBS + 1% BSA, sterile-filtered and frozen at -70 'C until further use. Examination material:
• Glycoproteins secreted glycoproteins such as mucus or plasma proteins are particularly suitable for immobilization on microtiter plates. These connections are easily accessible and can be removed from the patient during routine examinations (endoscopy, puncture). Fresh or freeze-dried material can be used for the test. For glycoproteins that have to be detached from the plasma membrane with the aid of detergents, nitrocellulose is more suitable as a carrier material (see below).
• Gangliosides These glycolipids are components of the cytoplasmic membrane and are extracted from small tissue samples (approx. 1 g) with organic solvents.
• Freeze-dried glycoproteins are dissolved in PBS and 100 ⁇ l of this solution are pipetted into each well.
• the adhesion er ⁇ follows h overnight at 4 'C, or at room temperature for 1-2.
• Ganglipsides are dissolved in methanol and 100 ⁇ l of this solution are added to each well. The solvent volatilizes at room temperature. Adherent growing cells (cell culture) are washed 3 times with PBS (test in small culture dishes).
• Non-adherent cells are suspended at 1-2% in PBS and 100 ⁇ l each in those pretreated with poly-1-lysine
• Positive controls eg rat serum, bovine submandibular mucin, bovine brain gangliosides.
• Test 2 eg rat serum, bovine submandibular mucin, bovine brain gangliosides.
• Cells or complex carbohydrates are not bound directly to the solid phase but with the aid of antibodies, lectins or cells which have not previously been adsorbed onto the carrier material and which themselves do not have 9-N / O-acetylated sialic acids
• O-acetylated gangliosides on thin-layer plates using precipitating, chromogenic substrates (e.g. ⁇ -naphthyl acetate).
• Thin-film panels e.g. B. HPTL silica gel plates on aluminum; 5 x 7.5 cm, layer thickness 0.2 mm (Merck, Darmstadt). Corresponding plates on glass or optionally cellulose plates can also be used.
• Fixative 0.5% polyisobutyl ethacrylate in diethyl ether
• step 1 Halve the thin-layer plate with samples carried in parallel after step 2. One half is sprayed with orcinol reagent and so all sialic acid-containing compounds are made visible; the other half is described in step 3-10.

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• 1991
  • 1991-06-10 DE DE19914119085 patent/DE4119085A1/de active Granted
• 1992
  • 1992-06-10 EP EP92912384A patent/EP0589983A1/de not_active Ceased
  • 1992-06-10 WO PCT/EP1992/001296 patent/WO1992022672A1/de not_active Ceased

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