EP1141721A1 - Conjugues pour deceler la presence de virus provoquant des maladies des voies respiratoires, et leur utilisation - Google Patents

Conjugues pour deceler la presence de virus provoquant des maladies des voies respiratoires, et leur utilisation

Info

Publication number
EP1141721A1
EP1141721A1 EP99965481A EP99965481A EP1141721A1 EP 1141721 A1 EP1141721 A1 EP 1141721A1 EP 99965481 A EP99965481 A EP 99965481A EP 99965481 A EP99965481 A EP 99965481A EP 1141721 A1 EP1141721 A1 EP 1141721A1
Authority
EP
European Patent Office
Prior art keywords
virus
influenza
conjugate according
viruses
conjugate
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Withdrawn
Application number
EP99965481A
Other languages
German (de)
English (en)
Inventor
Georg Opitz
Hartwig Höcker
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Diagor GmbH
Original Assignee
Diagor GmbH
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Diagor GmbH filed Critical Diagor GmbH
Priority to EP99965481A priority Critical patent/EP1141721A1/fr
Publication of EP1141721A1 publication Critical patent/EP1141721A1/fr
Withdrawn legal-status Critical Current

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

Landscapes

  • 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

L'invention concerne un conjugué constitué d'un vecteur à haut poids moléculaire, sans risque toxicologique, et d'une substance se liant spécifiquement aux virus. L'invention concerne également l'utilisation dudit conjugué et un procédé pour enrichir des virus et pour déceler, simultanément ou ultérieurement, leur présence. L'invention concerne en particulier un procédé pour enrichir des virus grippaux A/B et pour déceler leur présence.
EP99965481A 1998-12-18 1999-12-16 Conjugues pour deceler la presence de virus provoquant des maladies des voies respiratoires, et leur utilisation Withdrawn EP1141721A1 (fr)

Priority Applications (1)

Application Number Priority Date Filing Date Title
EP99965481A EP1141721A1 (fr) 1998-12-18 1999-12-16 Conjugues pour deceler la presence de virus provoquant des maladies des voies respiratoires, et leur utilisation

Applications Claiming Priority (4)

Application Number Priority Date Filing Date Title
EP98124131 1998-12-18
EP98124131A EP1010981A1 (fr) 1998-12-18 1998-12-18 Conjugés pour détection de virus et leur utilisation
PCT/EP1999/010021 WO2000037942A1 (fr) 1998-12-18 1999-12-16 Conjugues pour deceler la presence de virus provoquant des maladies des voies respiratoires, et leur utilisation
EP99965481A EP1141721A1 (fr) 1998-12-18 1999-12-16 Conjugues pour deceler la presence de virus provoquant des maladies des voies respiratoires, et leur utilisation

Publications (1)

Publication Number Publication Date
EP1141721A1 true EP1141721A1 (fr) 2001-10-10

Family

ID=8233180

Family Applications (2)

Application Number Title Priority Date Filing Date
EP98124131A Withdrawn EP1010981A1 (fr) 1998-12-18 1998-12-18 Conjugés pour détection de virus et leur utilisation
EP99965481A Withdrawn EP1141721A1 (fr) 1998-12-18 1999-12-16 Conjugues pour deceler la presence de virus provoquant des maladies des voies respiratoires, et leur utilisation

Family Applications Before (1)

Application Number Title Priority Date Filing Date
EP98124131A Withdrawn EP1010981A1 (fr) 1998-12-18 1998-12-18 Conjugés pour détection de virus et leur utilisation

Country Status (3)

Country Link
EP (2) EP1010981A1 (fr)
AU (1) AU2097900A (fr)
WO (1) WO2000037942A1 (fr)

Families Citing this family (5)

* Cited by examiner, † Cited by third party
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
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
DE102009015740A1 (de) * 2009-03-31 2010-10-14 Siemens Aktiengesellschaft Formmasse zur Erkennung der Krankheitserreger im Mund
WO2018050429A1 (fr) * 2016-09-19 2018-03-22 Julius-Maximilians-Universität Würzburg Détecteur pour diagnostic et gomme à mâcher comprenant ce détecteur pour diagnostic pour détecter des virus par le goût

Family Cites Families (4)

* Cited by examiner, † Cited by third party
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
JPS6147186A (ja) * 1984-08-09 1986-03-07 Chemo Sero Therapeut Res Inst インフルエンザウイルスの精製方法
JPH0614047B2 (ja) * 1987-02-27 1994-02-23 イーストマン コダック カンパニー 凝集反応試薬及びその製造方法
DE4243491A1 (de) * 1992-12-22 1994-06-23 Behringwerke Ag Verfahren zur Reinigung und Anreicherung von Rubella-Virus

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
See references of WO0037942A1 *

Also Published As

Publication number Publication date
EP1010981A1 (fr) 2000-06-21
AU2097900A (en) 2000-07-12
WO2000037942A1 (fr) 2000-06-29

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