WO2000037942A1 - Konjugate zum nachweis von atemwegserkrankung verursachenden viren und deren verwendung - Google Patents
Konjugate zum nachweis von atemwegserkrankung verursachenden viren und deren verwendung Download PDFInfo
- Publication number
- WO2000037942A1 WO2000037942A1 PCT/EP1999/010021 EP9910021W WO0037942A1 WO 2000037942 A1 WO2000037942 A1 WO 2000037942A1 EP 9910021 W EP9910021 W EP 9910021W WO 0037942 A1 WO0037942 A1 WO 0037942A1
- Authority
- WO
- WIPO (PCT)
- Prior art keywords
- virus
- influenza
- conjugate according
- viruses
- conjugate
- Prior art date
Links
Classifications
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N7/00—Viruses; Bacteriophages; Compositions thereof; Preparation or purification thereof
-
- 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
- G01N33/531—Production of immunochemical test materials
- G01N33/532—Production of labelled immunochemicals
-
- 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
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N2760/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA ssRNA viruses negative-sense
- C12N2760/00011—Details
- C12N2760/16011—Orthomyxoviridae
- C12N2760/16111—Influenzavirus A, i.e. influenza A virus
- C12N2760/16151—Methods of production or purification of viral material
Definitions
- the invention relates to a conjugate consisting of a toxicologically harmless high molecular weight carrier and a specific virus-binding substance, its use and a method for the enrichment and subsequent or simultaneous detection of viruses.
- the invention relates in particular to a method for the enrichment and diagnosis of influenza A / B viruses
- Viruses in the nose, mouth and throat often lead to diseases and are not easy to detect. This applies in particular to influenza A / B viruses
- Influenza A / B viruses are responsible for the flu, which affects several hundred million people worldwide every year. Influenza A / B virus-related flu is difficult to distinguish from other acute respiratory infections. This is only possible with influenza A / B virus-specific detection methods. A particular problem, however, is that the affected areas, ie. H. Throat and nose to get material that contains sufficient amounts of influenza A / B virus for detection. Corresponding samples are usually obtained in the form of smears, washes or aspirates. These samples can only be taken by trained personnel. The virus-containing material obtained must be laboriously cleaned, e.g.
- the present invention describes a simple way of isolating and detecting viruses in sufficient quantities.
- Viruses in the sense of the invention are in particular viruses of the throat, mouth, throat and nose. These are influenza A and influenza B viruses, respiratory syncytial virus, parainfluenza viruses, Rhinoviruses and adenoviruses Viruses to be detected are influenza A or B
- the viruses are enriched by conjugates consisting of a toxicologically harmless high molecular weight carrier and a virus binding substance.
- the high molecular weight carriers are in particular a toxicologically harmless polymer with coupling-capable groups, selected from the group consisting of natural rubber, silicone rubber and polyolefins and the derivatives of these polymers.
- the conjugates Used in the form of chewing gum or lollipop to bind and enrich virus from the saliva of patients. It is also possible to enrich the virus contained in the rinsing water of a mouthwash by gargling with corresponding conjugate particles ex vivo.
- These particles are spherical, for example, especially small macroscopic spheres (diameter between 500 ⁇ m up to 5 mm) These particles can alternatively be added to the gargle water for in vivo enrichment.
- the virus bound to the polymer conjugate is directly via its enzymatic Activity or determined after desorption using an in vitro rapid virus test Various in vitro rapid tests are generally accessible
- Influenza A / B virus is released by infected cells of the respiratory tract. One part of the virus remains on the surface of the cells, the other part gets into the saliva or nasal secretion, which is ultimately responsible for the fact that the formation of small influenza A / B -Virus-containing droplets on exhalation, but especially by coughing or sneezing, the virus is spread (droplet infection)
- specific influenza A / B virus-binding molecules With the help of specific influenza A / B virus-binding molecules, it is possible to find influenza A / B virus in the throat and mouth to bind, enrich and diagnose
- the specific influenza A / B virus-binding molecules are chemically coupled to toxicologically harmless, water-insoluble polymers. Preferred are covalent conjugates.
- Neuraminic acid and its derivatives, in particular ⁇ -2-O, have proven to be suitable for this purpose -methyl-5-N-thioacetylneuraminic acid, and neuraminidase inhibitors of the type zanamivir (4-guanidinoNeu5Acen) or GS 4071 ((3R, 4R, 5S ) -4-acetamido-5-amino-3- (l-ethylpropoxy) -l-cyclohexane-carboxylic acid) or specific antibodies against influenza A / B virus, especially monoclonal antibodies Suitable binding partners are polymers that are non-toxic in all respects and that have rubber-elastic properties on the one hand.
- polystyrene foam An obvious example is the natural rubber used in chewing gum. More suitable is silicone rubber, which is better defined and its properties can be better adjusted by crosslinking.
- polyolefins are also in shape of soft foam can be used. They have a particularly large surface area.
- the polymers mentioned are to be regarded as examples. Basically, by appropriate modification, almost all polymers can be adjusted in such a way that they are characterized by rubber-elastic properties and are retained even during use
- high-molecular carrier materials with a high surface area and / or high coverage of the virus-binding substance must be used.
- the conjugate is then used, for example, in the form of a swab or thin elastic rod
- the conjugate is used in the oral cavity, bite and chewable high molecular weight carrier material is preferred.
- the molecule specifically binding the influenza A / B virus is to be immobilized on the surface of the above-mentioned rubber-elastic materials.
- the creation of functional groups on the surface of the polymer material is necessary This is done, for example, by treatment with a plasma to generate polymer-bound radicals which are able to initiate the radical polymerization of suitable monomers, for example acrylic acid.
- suitable monomers for example acrylic acid.
- functional groups in the present case carboxy groups
- Another possibility of binding the virus-binding substance to the high molecular weight carrier consists in the biotin / steptavidine coupling known to the person skilled in the art
- a molecule which is suitable for binding to the carboxyl groups of the modified rubber is, for example, ⁇ -2-O-methyl-5-N-thioacetylneuraminic acid which can be reacted with aminothioethanol to give the corresponding amine
- the density of the influenza A / B virus binding molecules on the surface of the polymer e.g. one
- Neuraminic acid derivative This can be adjusted by using an appropriate mixing ratio of acrylic acid with acrylamide. With increasing acrylamide content, the concentration of the carboxy groups on the polymer and thus the density of the neuraminic acid groups on the surface of the rubber-elastic polymer can be reduced.
- the polymer conjugate in the form of chewing gum or lollipop has proven to be most suitable for binding and enriching influenza A / B virus in the oral cavity.
- the lollipop or chewing gum is designed so that the virus-binding molecules are on its surface.
- the shape of a lollipop lends itself, since it makes it easy to obtain samples even from children - an important group of patients. Appropriate shaping means that the lollipop can be swallowed. If necessary, the rubber-elastic polymer of the lollipop could even be flavored for better acceptance in children.
- the material of the conjugate should be bite / chewable, because the increased salivation during chewing releases more viruses from the infected tissue of the throat. The increased virus density in the saliva leads to an increased accumulation of viruses on the conjugate.
- the influenza A B virus binding conjugate is exposed to saliva for up to 30 minutes, preferably up to 15 minutes, in particular about 3 to 7 minutes.
- the binding of the virus to the polymer conjugate depends on the incubation time and virus concentration. A 10 minute incubation period proves to be sufficient.
- suitable detergents e.g. Triton X-100
- the influenza A / B virus obtained with the polymer conjugate can be detected in vitro using one of the usual diagnostic influenza A / B tests.
- Influenza A / B virus can be detected immunologically, preferably by means of influenza A B-specific antibodies coupled with enzyme.
- Influenza AB virus can also be detected enzymatically, preferably via the neuraminidase activity of the virus in a chromogenic or fluorogenic test.
- the influenza A / B virus bound to the polymer conjugate can also be detected directly via the enzymatic activity of the neuraminidase characteristic of influenza AB virus.
- a chromogenic substrate of the neuraminidase is incubated with the polymer conjugate containing influenza A / B virus.
- One with the enzymatic cleavage of the chromogenic The accompanying color change indicates the presence of influenza A / B virus
- a polydimethylsiloxane film is treated under argon plasma under standard conditions for 15 seconds and then exposed to the air.
- the film is then transferred to the aqueous solution of acrylic acid and the graft copolymerization is initiated thermally or photochemically.
- the aqueous solution begins to become cloudy, the film is removed from the solution and rinsed with water Then it is reacted with an aqueous solution of EDC and finally reacted with glycol-2,2-diaminoethyl ether.
- the 3-acetylmercaptopropionic acid is reacted with an excess of the N-hydroxysuccinimide active ester. It is then washed and deacetylated with 25 mmolar hydroxylamine solution.
- the biochemically active agent is added with an N-terminal maleimide group and bound in the manner of a Michael addition
- a square centimeter of the polymer conjugate film produced in this way is immersed in vessels with 5 ml of nutrient solution which contain different concentrations of influenza A / B virus.
- the film is incubated for 10 minutes.
- the virus bound in the nutrient solution and on the polymer conjugate can be detected using a rapid influenza test.
- Via the polymer conjugate film the influenza A / B virus can still be detected in a concentration which is 10 times lower than that which can be detected with the test in the original nutrient solution.
- a ten-fold enrichment of the virus was achieved
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- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Immunology (AREA)
- Engineering & Computer Science (AREA)
- Chemical & Material Sciences (AREA)
- Biomedical Technology (AREA)
- Molecular Biology (AREA)
- Hematology (AREA)
- Urology & Nephrology (AREA)
- Microbiology (AREA)
- Biochemistry (AREA)
- Virology (AREA)
- General Health & Medical Sciences (AREA)
- Medicinal Chemistry (AREA)
- Biotechnology (AREA)
- Physics & Mathematics (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Food Science & Technology (AREA)
- General Physics & Mathematics (AREA)
- Pathology (AREA)
- Cell Biology (AREA)
- Organic Chemistry (AREA)
- Analytical Chemistry (AREA)
- Genetics & Genomics (AREA)
- Wood Science & Technology (AREA)
- Zoology (AREA)
- General Engineering & Computer Science (AREA)
- Tropical Medicine & Parasitology (AREA)
- Medicines Containing Antibodies Or Antigens For Use As Internal Diagnostic Agents (AREA)
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
- Investigating Or Analysing Biological Materials (AREA)
Abstract
Description
Claims
Priority Applications (2)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
AU20979/00A AU2097900A (en) | 1998-12-18 | 1999-12-16 | Conjugates for detecting viruses that cause diseases of the respiratory tract and use thereof |
EP99965481A EP1141721A1 (de) | 1998-12-18 | 1999-12-16 | Konjugate zum nachweis von atemwegserkrankung verursachenden viren und deren verwendung |
Applications Claiming Priority (2)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
EP98124131.8 | 1998-12-18 | ||
EP98124131A EP1010981A1 (de) | 1998-12-18 | 1998-12-18 | Konjugate zum Nachweis von Viren und deren Verwendung |
Publications (1)
Publication Number | Publication Date |
---|---|
WO2000037942A1 true WO2000037942A1 (de) | 2000-06-29 |
Family
ID=8233180
Family Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
PCT/EP1999/010021 WO2000037942A1 (de) | 1998-12-18 | 1999-12-16 | Konjugate zum nachweis von atemwegserkrankung verursachenden viren und deren verwendung |
Country Status (3)
Country | Link |
---|---|
EP (2) | EP1010981A1 (de) |
AU (1) | AU2097900A (de) |
WO (1) | WO2000037942A1 (de) |
Cited By (3)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
DE10155693A1 (de) * | 2001-11-07 | 2003-05-15 | Florian Schweigert | Verfahren zum Nachweis von apathogenen Partikeln und deren pathogenen Varianten im Organismus |
DE10155692A1 (de) * | 2001-11-07 | 2003-05-22 | Florian Schweigert | Verfahren zum Nachweis von endogenen und exogenen Stoffen im Organismus |
EP3516067B1 (de) * | 2016-09-19 | 2022-04-06 | Julius-Maximilians-Universität Würzburg | Verbindung und kaugummi umfassend die verbindung zum geschmacklichen nachweis von viren |
Families Citing this family (2)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
DE10028837A1 (de) | 2000-06-15 | 2001-12-20 | Roche Diagnostics Gmbh | Verfahren zum Nachweis von Influenza A/B-Viren |
DE102009015740A1 (de) * | 2009-03-31 | 2010-10-14 | Siemens Aktiengesellschaft | Formmasse zur Erkennung der Krankheitserreger im Mund |
Citations (4)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
JPS5772063A (en) * | 1980-10-23 | 1982-05-06 | Masafumi Mizogami | Detection method for hepatitis b virus e antigen |
EP0171086A2 (de) * | 1984-08-09 | 1986-02-12 | Juridical Foundation The Chemo-Sero-Therapeutic Research Institute | Verfahren zur Reinigung des Influenzavirus |
EP0280561A2 (de) * | 1987-02-27 | 1988-08-31 | EASTMAN KODAK COMPANY (a New Jersey corporation) | Agglutinationsreagens und Verfahren zu seiner Herstellung |
EP0603615A2 (de) * | 1992-12-22 | 1994-06-29 | BEHRINGWERKE Aktiengesellschaft | Verfahren zur Reinigung und Anreicherung von Rubella-Virus |
-
1998
- 1998-12-18 EP EP98124131A patent/EP1010981A1/de not_active Withdrawn
-
1999
- 1999-12-16 AU AU20979/00A patent/AU2097900A/en not_active Abandoned
- 1999-12-16 WO PCT/EP1999/010021 patent/WO2000037942A1/de not_active Application Discontinuation
- 1999-12-16 EP EP99965481A patent/EP1141721A1/de not_active Withdrawn
Patent Citations (4)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
JPS5772063A (en) * | 1980-10-23 | 1982-05-06 | Masafumi Mizogami | Detection method for hepatitis b virus e antigen |
EP0171086A2 (de) * | 1984-08-09 | 1986-02-12 | Juridical Foundation The Chemo-Sero-Therapeutic Research Institute | Verfahren zur Reinigung des Influenzavirus |
EP0280561A2 (de) * | 1987-02-27 | 1988-08-31 | EASTMAN KODAK COMPANY (a New Jersey corporation) | Agglutinationsreagens und Verfahren zu seiner Herstellung |
EP0603615A2 (de) * | 1992-12-22 | 1994-06-29 | BEHRINGWERKE Aktiengesellschaft | Verfahren zur Reinigung und Anreicherung von Rubella-Virus |
Non-Patent Citations (1)
Title |
---|
PATENT ABSTRACTS OF JAPAN vol. 6, no. 152 (P - 134) 12 August 1982 (1982-08-12) * |
Cited By (3)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
DE10155693A1 (de) * | 2001-11-07 | 2003-05-15 | Florian Schweigert | Verfahren zum Nachweis von apathogenen Partikeln und deren pathogenen Varianten im Organismus |
DE10155692A1 (de) * | 2001-11-07 | 2003-05-22 | Florian Schweigert | Verfahren zum Nachweis von endogenen und exogenen Stoffen im Organismus |
EP3516067B1 (de) * | 2016-09-19 | 2022-04-06 | Julius-Maximilians-Universität Würzburg | Verbindung und kaugummi umfassend die verbindung zum geschmacklichen nachweis von viren |
Also Published As
Publication number | Publication date |
---|---|
AU2097900A (en) | 2000-07-12 |
EP1010981A1 (de) | 2000-06-21 |
EP1141721A1 (de) | 2001-10-10 |
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