EP1141721A1 - Conjugates for detecting viruses that cause diseases of the respiratory tract and use thereof - Google Patents

Conjugates for detecting viruses that cause diseases of the respiratory tract and use thereof

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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
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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.)
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Application number
EP99965481A
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German (de)
French (fr)
Inventor
Georg Opitz
Hartwig Höcker
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Diagor GmbH
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Diagor GmbH
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Application filed by Diagor GmbH filed Critical Diagor GmbH
Priority to EP99965481A priority Critical patent/EP1141721A1/en
Publication of EP1141721A1 publication Critical patent/EP1141721A1/en
Withdrawn legal-status Critical Current

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    • 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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  • Life Sciences & Earth Sciences (AREA)
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  • Urology & Nephrology (AREA)
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  • Virology (AREA)
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  • Food Science & Technology (AREA)
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  • Genetics & Genomics (AREA)
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  • General Engineering & Computer Science (AREA)
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Abstract

The invention relates to a conjugate consisting of a toxicologically harmless high molecular weight carrier and a specific virus binding substance, the use thereof and a method for enriching and subsequent or simultaneous detection of viruses. The invention specifically relates to a method for enriching and diagnosing A/B influenza viruses.

Description

Konjugate zum Nachweis von Atemwegserkrankung verursachenden Viren und derenConjugates for the detection of viruses that cause respiratory diseases and their
Verwendunguse
Die Erfindung betrifft ein Konjugat bestehend aus einem toxikologisch unbedenklichen hochmolekularen Träger und einer spezifisch Virus-bindenden Substanz, dessen Verwendung und einem Verfahren zum Anreichern und anschließendem oder gleichzeitigen Nachweis von Viren. Die Erfindung betrifft im besonderen ein Verfahren zur Anreicherung und Diagnose von Influenza A/B VirenThe 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
Beschreibungdescription
Viren im Nase-, Mund- und Rachenraum fuhren häufig zu Erkrankungen und sind nicht einfach nachzuweisen Dies gilt insbesondere für Influenza A/B VirenViruses 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 Viren sind verantwortlich für die Grippe, an der jahrlich mehrere 100 Millionen Menschen weltweit erkranken. Die Influenza-A/B-Virus-bedingte Grippe laßt sich nur schwer von anderen akuten respiratorischen Infekten unterscheiden Möglich ist dies nur mit Influenza-A/B-Virus-spezifischen Nachweisverfahren Ein besonderes Problem besteht indes darin, aus den befallenen Bereichen, d. h. Rachen und Nase, Material zu bekommen, das für einen Nachweis ausreichende Mengen an Influenza A/B Virus enthält. Normalerweise werden entsprechende Proben in Form von Abstrichen, Waschungen oder Aspiraten gewonnen Diese Entnahmen von Proben sind nur von geschultem Personal durchführbar Das gewonnene virushaltige Material muß aufwendig aufgereinigt werden, z.B durch Ultrazentrifugation oder differentielle Zentrifugation im Dichtegradienten, wobei entweder der Virus alleine oder der Virus an Erythrozyten gebunden (z.B. EP 603 615) isoliert wird. Aus EP 280 561 sind noch andere mikroskopische Partikel bekannt, die zur Virusanreicherung verwendet werden Die Isolation des Virus durch die genannten Verfahren verlangt geschultes Personal, da die virushaltigen mikroskopischen Partikel nur aufwendig zu isolieren sind Auch Verfahren zur Virusaufreinigung mittels Säulenchromatographie, wie in EP 171 086 beschrieben, sind nur mit geschultes Personal möglich Wünschenswert wäre ein Verfahren zur Gewinnung von Influenza- A/B-Virus-haltigem Probenmaterial und deren Nachweis durch den Patienten selbstInfluenza 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. by ultracentrifugation or differential centrifugation in density gradient, with either the virus alone or the virus Erythrocytes bound (eg EP 603 615) is isolated. Other microscopic particles which are used for virus enrichment are known from EP 280 561. The isolation of the virus by the above-mentioned methods requires trained personnel, since the virus-containing microscopic particles are difficult to isolate. Methods for virus purification by means of column chromatography, as in EP 171 086 described, are only possible with trained personnel. A method for obtaining sample material containing influenza A / B virus and its detection by the patient himself would be desirable
Die vorliegende Erfindung beschreibt eine einfache Möglichkeit Viren in ausreichenden Mengen zu isolieren und nachzuweisen Als Viren im Sinne der Erfindung gelten insbesondere Viren des Halses, Mundes, Rachen und der Nase. Dies sind Influenza A und Influenza B Viren, Respiratorischer Syncytialvirus, Parainfluenza Viren, Rhinoviren und Adenoviren Bevorzugt nachzuweisende Viren sind Influenza A oder BThe 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
VirenViruses
Die Viren werden durch Konjugate bestehend aus einem toxikologisch unbedenklichem hochmolekularen Trager und einer Viren bindenden Substanz angereichert Die hochmolekularen Trager sind insbesondere ein toxikologisch unbedenkliches Polymer mit kopplungsfahigen Gruppen, ausgewählt aus der Gruppe bestehend aus Naturkautschuk, Silikonkautschuk und Polyolefine und die Derivate dieser Polymere Die Konjugate werden in Form von Kaugummi oder Lutscher zur Bindung und Anreicherung von Virus aus dem Speichel von Patienten verwendet Ebenso ist es möglich, das im Spulwasser einer Mundspulung durch Gurgeln enthaltene Virus mit entsprechenden Konjugatpartikeln ex vivo anzureichern Diese Partikel sind beispielsweise sphärisch, insbesondere kleine makroskopische Kugelchen (Durchmesser zwischen 500μm bis zu 5 mm) Diese Partikel können alternativ auch schon dem Gurgelwasser zugesetzt werden für eine in vivo Anreicherung Das an das Polymerkonjugat gebundene Virus wird direkt über seine enzymatische Aktivität oder nach Desorption mit einem in vitro Virusschnelltest bestimmt Es sind verschiedene in vitro Schnelltests allgemein zuganglichThe 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 500μm 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
Im folgenden wird die Erfindung am Beispiel des Influenza A/B Virus erläutert Der Fachmann kann die erfindungsgemaßen Ausführungsformen an andere Viren anpassenThe invention is explained below using the example of the influenza A / B virus. The person skilled in the art can adapt the embodiments according to the invention to other viruses
Influenza A/B Virus wird von infizierten Zellen des Atmungstraktes freigesetzt Ein Teil des Virus verbleibt auf der Oberflache der Zellen, der andere Teil gelangt in den Speichel bzw das Nasensekret, was letztlich dafür verantwortlich ist, daß über die Bildung kleiner Influenza- A/B -Virus-haltiger Tropfchen beim Ausatmen, vor allem aber durch Husten oder Niesen, das Virus verbreitet wird ( Tropfcheninfektion ) Mit Hilfe spezifischer Influenza-A/B-Virus-bindender Moleküle ist es möglich, im Rachen und Mund befindliches Influenza A/B Virus zu binden, anzureichern und zu diagnostizieren Dazu werden an toxikologisch unbedenkliche, wasserunlösliche Polymere die spezifisch Influenza- A/B-Virus-bindenden Moleküle chemisch gekoppelt Bevorzugt sind kovalente Konjugate Als dafür geeignet erwies sich u a Neuraminsaure und deren Derivate, insbesondere α-2-O-methyl-5-N- thioacetylneuraminsaure, sowie Neuraminidase Inhibitoren vom Typ Zanamivir (4- guanidinoNeu5Acen) oder GS 4071 ((3R,4R,5S)-4-acetamido-5-amino-3-(l- ethylpropoxy)-l-cyclohexane-carboxylsaure) oder spezifische Antikörper gegen Influenza A/B Virus, insbesondere monoklonale Antikörper Als Bindungspartner sind Polymere geeignet, die einerseits in jeder Beziehung ungiftig sind und andererseits kautschukelastische Eigenschaften besitzen Ein naheliegendes Beispiel ist der in Kaugummis benutzte Naturkautschuk Geeigneter ist Silikonkautschuk, der besser definiert und durch Vernetzung hinsichtlich seiner Eigenschaften besser einstellbar ist Darüber hinaus sind auch Polyolefine in Form von Weichschaum einsetzbar Sie weisen eine besonders große Oberflache auf Die erwähnten Polymere sollen als Beispiele gelten, grundsatzlich lassen sich durch entsprechende Modifizierung nahezu alle Polymere so einstellen, daß sie durch kautschukelastische Eigenschaften gekennzeichnet sind und diese auch wahrend des Gebrauchs beibehaltenInfluenza 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) 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 For this purpose, 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 α-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. 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. In addition, 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
Bei der Probenentnahme in der Nase müssen hochmolekulare Tragermaterialien mit hoher Oberflache und/oder hoher Belegung der Virus-bindenden Substanz verwendet werden Das Konjugat wird dann zum Beispiel in Form eines Tupfers oder dünnen elastischen Stabes verwendetWhen taking samples in the nose, 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
Wird das Konjugat im Mundraum verwendet, so ist biß- und kaustabiles hochmolekulares Tragermaterial bevorzugt Das Influenza A/B Virus spezifisch bindende Molekül ist auf der Oberflache der oben genannten kautschukelastischen Materialien zu immobilisieren Hierzu ist zunächst die Schaffung von funktioneilen Gruppen auf der Oberflache des Polymermaterials erforderlich Dies geschieht z B durch Behandlung mit einem Plasma zur Erzeugung von Polymer-gebundenen Radikalen, die in der Lage sind, die radikalische Polymerisation geeigneter Monomerer, z B von Acrylsäure zu initiieren Auf diese Weise werden auf der Oberflache f nktionelle Gruppen, im vorliegenden Fall Carboxygruppen erzeugt, die nach Methoden der Peptidchemie mit Aminogruppen zu Amidbindungen umgesetzt werden können Eine weitere Möglichkeit der Bindung der virusbindenden Substanz an den hochmolekularen Trager besteht in der dem Fachmann bekannten Biotin/SteptavidinkopplungIf 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. First of all, 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. In this way, functional groups, in the present case carboxy groups, are formed on the surface generated, which can be converted with amino groups to amide bonds using methods of peptide chemistry. 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
Als Molekül, das sich zur Anbindung an die Carboxylgruppen des modifizierten Kautschuks eignet, ist z B α-2-O-methyl-5-N-thioacetylneuraminsaure anzusehen, das mit Aminothioethanol zu dem entsprechenden Amin umgesetzt werden kannA molecule which is suitable for binding to the carboxyl groups of the modified rubber is, for example, α-2-O-methyl-5-N-thioacetylneuraminic acid which can be reacted with aminothioethanol to give the corresponding amine
Alternativ lassen sich die Carboxylgruppen der auf der Oberflache gepfropften Polyacrylsaure unter Anwendung der Cabodiimidmethode (EDC) mit Glycol-2,2- diaminodiethy lether als Spacer umsetzen, wobei eine Aminogruppe für weitere Reaktionen frei bleibt, z B mit dem N-Hydroxysuccinimid-Aktivester, der 3- Acetylmercatopropionsaure Nach Deacetylierung mit Hydroxylamin kann die Mercaptogruppe im Sinne einer Michael-Addition an die Doppelbindung derAlternatively, the carboxyl groups of the polyacrylic acid grafted on the surface can be reacted using the cabodiimide method (EDC) with glycol-2,2-diaminodiethyl ether as a spacer, an amino group remaining free for further reactions, for example with the N-hydroxysuccinimide active ester, 3-acetylmercatopropionic acid After deacetylation with hydroxylamine, the Mercapto group in the sense of a Michael addition to the double bond of
Maleinimidgruppe, die an den Wirkstoff gebunden ist, addiert werden.Maleimide group, which is bound to the active ingredient, are added.
Von wesentlicher Bedeutung ist die Dichte der auf der Oberfläche des Polymers befindlichen Influenza-A/B-Virus-bindenden Moleküle, z.B. einesThe density of the influenza A / B virus binding molecules on the surface of the polymer, e.g. one
Neuraminsäurederivats. Diese läßt sich einstellen, indem ein entsprechendes Mischungsverhältnis von Acrylsäure mit Acrylamid eingesetzt wird. Mit zunehmenden Acrylamidanteil kann die Konzentration der Carboxygruppen auf dem Polymer und damit die Dichte der Neuraminsäuregruppen auf der Oberfläche des kautschukelastischen Polymers reduziert werden.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.
Zur Bindung und Anreicherung von Influenza A/B Virus im Mundraum erweist sich das Polymerkonjugat in Form von Kaugummi bzw. Lutscher als am besten geeignet. Der Lutscher bzw. das Kaugummi ist so gestaltet, daß die Virus-bindenden Moleküle sich auf ihrer Oberfläche befinden. Die Form eines Lutscher bietet sich an, da damit auch bei Kindern - einer wichtigen Patientengruppe - eine Probengewinnung problemlos möglich ist. Durch entsprechende Formgebung läßt sich ausschließen, daß der Lutscher verschluckt werden kann. Falls erforderlich, ließe sich sogar zur besseren Akzeptanz bei Kindern das kautschukelastische Polymer des Lutschers mit Geschmackstoffen versehen. Das Material des Konjugats sollte beiß/kaufähig sein, da über die gesteigerte Speichelbildung beim Kauen mehr Viren vom infizierten Gewebe des Rachenraums freigesetzt werden. Die so im Speichel erhöhte Virusdichte fuhrt zu einer verstärkten Anreicherung von Viren auf dem Konjugat. Das Influenza-A B-Virus-bindende Konjugat wird bis zu 30 Minuten dem Speichel ausgesetzt, bevorzugt bis zu 15 Minuten, insbesondere etwa 3 bis 7 Minuten.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.
Die Bindung des Virus an das Polymerkonjugat ist abhängig von der Inkubationszeit und Viruskonzentration. Eine 10 minütige Inkubationzeit erweist sich als ausreichend. Nachdem das Virus vom Polymerkonjugat durch geeignete Detergentien (z.B. Triton X- 100) gelöst und aufgeschlossen wurde, läßt sich das mit Polymerkonjugat gewonnene Influenza A/B Virus mit einem der üblichen diagnostischen Influenza A/B in vitro Schnelltests nachweisen.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. After the virus has been detached from the polymer conjugate by suitable detergents (e.g. Triton X-100) and digested, 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.
Der Nachweis von Influenza A/B Virus kann immunologisch erfolgen, bevorzugt mittels Influenza-A B-spezifischen, mit Enzym gekoppelten Antikörpern.Influenza A / B virus can be detected immunologically, preferably by means of influenza A B-specific antibodies coupled with enzyme.
Der Nachweis von Influenza A B Virus kann außerdem e zymatisch erfolgen, bevorzugt über die Neuraminidaseaktivität des Virus in einem chromogenen oder fluorogenen Test. Das auf dem Polymerkonjugat gebundene Influenza A/B Virus kann auch direkt über die enzymatische Aktivität der für Influenza A B Virus charakteπstischen Neuraminidase nachgewiesen werden Dazu wird ein chromogenes Substrat der Neuraminidase mit dem Influenza A/B Virus haltigen Polymerkonjugat inkubiert Eine mit der enzymatischen Spaltung des chromogenen Substrates einher gehende Farbanderung zeigt die Anwesenheit von Influenza A/B Virus anInfluenza 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. For this purpose, 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
Beispielexample
Eine Polydimethylsiloxan-Folie wird unter Standardbedingungen 15 sec im Argonplasma behandelt und anschließend der Luft ausgesetzt Daraufhin wird die Folie in die wäßrige Losung von Acrylsaure überführt und die Propfkopolymerisation thermisch oder photochemisch initiiert Wenn sich die wäßrige Losung zu trüben beginnt, wird die Folie der Losung entnommen und mit Wasser gespult Anschließend wird mit wäßriger Losung von EDC umgesetzt und schließlich mit Glycol-2,2- diaminoethylether umgesetzt Nach Spülen wird mit einem Überschuß des N- Hydroxysuccinimidaktivesters der 3-Acetylmercaptopropionsaure umgesetzt. Anschließend wird gewaschen und mit 25 mmolarer Hydroxylaminlosung deacetyliert Schließlich wird das biochemisch aktive Agens mit einer N-terminalen Maleinsaureimidgruppe hinzugefügt und im Sinne einer Michael-Addition gebundenA 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. When 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. After rinsing, 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. Finally, the biochemically active agent is added with an N-terminal maleimide group and bound in the manner of a Michael addition
Ein Quadratzentimeter der so hergestellten Polymerkonjugatfolie wird in Gefäße mit 5ml Nährlösung getaucht, die verschiedene Konzentrationen von Influenza A/B Virus enthalten Die Folie wird 10 Minuten lang inkubiert Mit einem Influenzaschnelltest laßt sich das in der Nährlösung und an dem Polymerkonjugat gebundene Virus nachweisen Über die Polymerkonjugatfolie läßt sich das Influenza A/B Virus in einer Konzentration noch nachweisen, die 10-fach niedriger ist als die, die sich in der ursprunglichen Nährlösung mit dem Test nachweisen laßt Mittels der Polymerkonjugatfolie ließ sich damit eine zehnfache Anreicherung des Virus erreichen 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. With the polymer conjugate film, a ten-fold enrichment of the virus was achieved

Claims

Ansprüche Expectations
1. Konjugat aus einem toxikologisch unbedenklichen hochmolekularen Träger und einer Viren bindenden Substanz, wobei der Virus ausgewählt ist aus der Gruppe bestehend aus Influenza A Virus, Influenza B Virus, Respiratorischer Syncytialvirus,1. conjugate of a toxicologically harmless high-molecular carrier and a virus-binding substance, the virus being selected from the group consisting of influenza A virus, influenza B virus, respiratory syncytial virus,
Parainfluenza Virus, Rhinovirus und Adenovirus.Parainfluenza virus, rhinovirus and adenovirus.
2. Konjugat nach Anspruch 1, wobei der hochmolekulare Träger ein toxikologisch unbedenkliches Polymer mit kopplungsfähigen Gruppen, ausgewählt aus der Gruppe bestehend aus Naturkautschuk, Silikonkautschuk und Polyolefine und deren Derivaten ist.2. Conjugate according to claim 1, wherein the high molecular weight carrier is a toxicologically acceptable polymer with groups capable of coupling, selected from the group consisting of natural rubber, silicone rubber and polyolefins and their derivatives.
3. Konjugat nach einem der Ansprüche 1 - 2, wobei die Viren bindende Substanz ausgewählt ist aus der Gruppe bestehend aus: Virus spezifische Antikörper, Neuraminsäure und deren Derivate, Inhibitoren der Influenza Neuraminidase.3. Conjugate according to one of claims 1-2, wherein the virus-binding substance is selected from the group consisting of: virus-specific antibodies, neuraminic acid and its derivatives, inhibitors of influenza neuraminidase.
4. Konjugat nach einem der Ansprüche 1 - 3, wobei die Viren bindende Substanz mit dem Polymer kovalent verbunden ist.4. Conjugate according to one of claims 1-3, wherein the virus-binding substance is covalently linked to the polymer.
5. Konjugat nach einem der Ansprüche 1 - 3, wobei die Viren bindende Substanz über Biotin/Streptavidin an das Polymer gebunden ist.5. Conjugate according to one of claims 1-3, wherein the virus-binding substance is bound to the polymer via biotin / streptavidin.
6. Mittel geeignet zum Anreichern eines Virus im Mund, Rachen oder in der Nase enthaltend ein Konjugat nach einem der Ansprüche 1 - 5. 6. Agent suitable for enriching a virus in the mouth, throat or nose containing a conjugate according to any one of claims 1-5.
7. Mittel nach Anspruch 6 in Form eines Lutschers, Kaugummis oder sphärischen, makroskopischen Partikeln.7. Composition according to claim 6 in the form of a lollipop, chewing gum or spherical, macroscopic particles.
8. Komplex bestehend aus einem Konjugat nach einem der Ansprüche 1 - 5 und dem anzureichernden Virus.8. Complex consisting of a conjugate according to one of claims 1-5 and the virus to be enriched.
9. Verwendung eines Konjugats nach einem der Ansprüche 1 - 5 zur Herstellung eines Diagnostikums zum Nachweis von Viren in Mund, Rachen oder Nase.9. Use of a conjugate according to any one of claims 1-5 for the manufacture of a diagnostic agent for the detection of viruses in the mouth, throat or nose.
10. Verfahren zum Nachweis von Viren, umfassend die Schritte: a. in vivo Anreicherung der Virus durch ein Konjugat nach einem der Ansprüche 1 - 5 b. Nachweis des Virus in vitro i) durch enzymatische Aktivität des gebundenen Virus oder ii) nach Desorption des Virus mit anschließendem oder gleichzeitigem separaten Nachweistest für den Virus. 10. A method for detecting viruses, comprising the steps of: a. in vivo enrichment of the virus by a conjugate according to one of claims 1-5 b. Detection of the virus in vitro i) by enzymatic activity of the bound virus or ii) after desorption of the virus with subsequent or simultaneous separate detection test for the virus.
EP99965481A 1998-12-18 1999-12-16 Conjugates for detecting viruses that cause diseases of the respiratory tract and use thereof Withdrawn EP1141721A1 (en)

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EP98124131A EP1010981A1 (en) 1998-12-18 1998-12-18 Conjugates to detect viruses and their use
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DE10028837A1 (en) 2000-06-15 2001-12-20 Roche Diagnostics Gmbh Detecting infection by influenza A or B virus, comprises rapid analysis of a saliva sample, with detection of nucleic acid or nucleoprotein
DE10155693A1 (en) * 2001-11-07 2003-05-15 Florian Schweigert Method for the detection of apathogenic particles and their pathogenic variants in the organism
DE10155692A1 (en) * 2001-11-07 2003-05-22 Florian Schweigert Procedure for the detection of endogenous and exogenous substances in the organism
DE102009015740A1 (en) * 2009-03-31 2010-10-14 Siemens Aktiengesellschaft Molded mass, where the surface of the molded mass is so provided or modified that an organic material is separated from aqueous saliva and bounded on surface, useful for detecting pathogens, preferably bacteria and viruses in mouth
WO2018050429A1 (en) * 2016-09-19 2018-03-22 Julius-Maximilians-Universität Würzburg Diagnostic sensor and chewing gum comprising such a diagnostic sensor for the taste-based detection of viruses

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JPS5772063A (en) * 1980-10-23 1982-05-06 Masafumi Mizogami Detection method for hepatitis b virus e antigen
JPS6147186A (en) * 1984-08-09 1986-03-07 Chemo Sero Therapeut Res Inst Method of purifying influenza virus
JPH0614047B2 (en) * 1987-02-27 1994-02-23 イーストマン コダック カンパニー Agglutination reagent and method for producing the same
DE4243491A1 (en) * 1992-12-22 1994-06-23 Behringwerke Ag Process for the purification and enrichment of Rubella virus

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