EP0707635A1 - Procede de depistage direct et biochimiquement fonctionnel de retrovirus dans des echantillons biologiques - Google Patents

Procede de depistage direct et biochimiquement fonctionnel de retrovirus dans des echantillons biologiques

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Publication number
EP0707635A1
EP0707635A1 EP94916144A EP94916144A EP0707635A1 EP 0707635 A1 EP0707635 A1 EP 0707635A1 EP 94916144 A EP94916144 A EP 94916144A EP 94916144 A EP94916144 A EP 94916144A EP 0707635 A1 EP0707635 A1 EP 0707635A1
Authority
EP
European Patent Office
Prior art keywords
retrovirus
virus
specific
reaction
carrier
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Withdrawn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
EP94916144A
Other languages
German (de)
English (en)
Inventor
Ortwin Faff
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.)
Individual
Original Assignee
Individual
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
Priority claimed from DE4416300A external-priority patent/DE4416300C2/de
Application filed by Individual filed Critical Individual
Publication of EP0707635A1 publication Critical patent/EP0707635A1/fr
Withdrawn legal-status Critical Current

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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
    • 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
    • C12Q1/48—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving transferase
    • 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
    • C12Q1/701—Specific hybridization probes
    • C12Q1/702—Specific hybridization probes for retroviruses
    • 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
    • C12Q1/701—Specific hybridization probes
    • C12Q1/702—Specific hybridization probes for retroviruses
    • C12Q1/703—Viruses associated with AIDS
    • 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
    • C12N2740/00—Reverse transcribing RNA viruses
    • C12N2740/00011—Details
    • C12N2740/10011—Retroviridae
    • C12N2740/10051—Methods of production or purification of viral material
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y10—TECHNICAL SUBJECTS COVERED BY FORMER USPC
    • Y10S—TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y10S435/00—Chemistry: molecular biology and microbiology
    • Y10S435/961—Chemistry: molecular biology and microbiology including a step of forming, releasing, or exposing the antigen or forming the hapten-immunogenic carrier complex or the antigen per se
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y10—TECHNICAL SUBJECTS COVERED BY FORMER USPC
    • Y10S—TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y10S435/00—Chemistry: molecular biology and microbiology
    • Y10S435/968—High energy substrates, e.g. fluorescent, chemiluminescent, radioactive
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y10—TECHNICAL SUBJECTS COVERED BY FORMER USPC
    • Y10S—TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y10S435/00—Chemistry: molecular biology and microbiology
    • Y10S435/974—Aids related test

Definitions

  • This analysis is based only on a non-functional or structure-specific molecular interaction between antibody and antigen or PCR primer and proviral DNA and therefore does not allow conclusions to be drawn about a whole and functional intact virus.
  • this analysis does not cover all stages of a viral infection, e.g. the phase in which the infection took place but no antibodies have yet been formed. These factors sometimes cause false positives or negatives
  • the direct and biochemically functional detection of retroviruses in biological samples is made possible by the present method, which consists of three stages: a) an extraction step, b) a reaction step and c) a detection step.
  • the extraction step is based on a selective binding of the retrovirus particles present in a sample on the basis of their surface molecules by means of specific ligands, which in turn are bound to a solid carrier material.
  • both virus particles and soluble viral surface molecules are immobilized in a carrier-ligand-virus complex and can be extracted selectively from the complex sample mixture.
  • a selective extraction of retroviral membrane-free virus core particles or intracisternal A particles using core-specific ligands is an alternative.
  • the selectivity of the extraction is that surface-specific ligands are used for the respective retrovirus species.
  • Virus-specific ligands can be: a) Antibodies
  • a retrovirus which are directed against antigenic surface epitopes of a retrovirus
  • c) anti-receptor idiotypic antibodies (monoclonal or polyclonal) that mimic the binding structures of the virus-specific receptors or d) complement -Proteins / peptides, which binds soluble virus-antibody-immune complexes existing in the sample via Ig-Fc regions.
  • a combination of several of the above-mentioned ligands can also be used for the selective binding of retrovirus particles.
  • the extraction step is based on purely structural interaction, it does not differentiate between whole virus particles and viral surface components e.g. Glycoproteins. Therefore, the carrier-ligand-virus complex is tested in a subsequent functional enzymatic reaction step, which is specific for retroviruses and can only be carried out by an intact virus particle, but not by individual virus components.
  • the reaction step is based on a retrovirus-specific enzymatic reaction which mimics the in vivo reactions in a retrovirus-infected cell and can be carried out by several specific viral enzymes: reverse transcriptase, RNAse H, integrase, protease and others. Endogenous reverse transcription is a complex process in which several viral components are involved, etc. the reverse transcription of the viral RNA (vRNA) to complementary DNA (cDNA) depending on the viral transfer RNA (tRNA), reverse transcriptase (RT) and exogenously added deoxy nucleotide triphosphate (dNTP s) functioning as a "primer".
  • vRNA viral RNA
  • cDNA complementary DNA
  • tRNA viral transfer RNA
  • RT reverse transcriptase
  • dNTP s exogenously added deoxy nucleotide triphosphate
  • the reverse transcription reaction can be continued. on the synthesis of the second cDNA strand or double-stranded cDNA (using DNA polymerase activity) and its subsequent integration into a plasmid (using retrovirus-specific integrase activity).
  • the integrated dsDNA like the previously synthesized cDNA, is detected in a subsequent detection step.
  • the enzymatically synthesized reaction products are quantitatively measured using radioactive or non-radioactive labeling using optical, fluorescence or luminescence methods.
  • the described method enables the routine direct and functional detection of retroviruses in biological samples for the first time. It has the following advantages over the existing detection methods: a) Retroviruses are detected with comparable sensitivity as particles specifically and directly in biological material using several components (antigens, enzymes and nucleic acids), b) the detection is not only carried out structurally (in the extraction step) more specific
  • retroviral T47D particles were used as the model virus, the properties typical of retrovirus: lipid membrane envelope, glycoproteins, reverse
  • the lysis reaction mix buffer had the following composition: 0.3% NP40, 50 mM Tris-HCl pH7.8, 40 mM KC1, 5mM MgCl 2 , 2mM DTT, 2mM EDTA and 30 ⁇ M dATP, dCTP, dGTP and dTTP + dUTP- Biotin + dUTP digoxigenin.
  • the incorporation of the dNTP ⁇ s or the synthesis of cDNA was then measured.
  • Detection The lysis-reaction mix was transferred to streptavidin-coated microtiter plates and incubated at 37 ° C. for 1 hour to immobilize the biotin and digoxigenin-labeled cDNA. After washing the unbound material, the immobilized cDNA was incubated with peroxidase-coupled anti-digoxigenin antibodies (1 hour at 37 ° C.), washed and then incubated with the peroxidase substrate ABTS (from Bschreibinger Mannheim). The color development was measured photometrically after 5 min at 405 nm (and 490 nm as reference) (Eberle, Land Seibl, B., J. Virol. Meth .: 40, 347-356, 1992). The results in Fig. 1 show that it is possible to immunologically extract T47D particles from cell culture medium with specific antibodies and then to demonstrate them biochemically by endogenous reverse transcription.

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  • Chemical & Material Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Health & Medical Sciences (AREA)
  • Organic Chemistry (AREA)
  • Zoology (AREA)
  • Wood Science & Technology (AREA)
  • Engineering & Computer Science (AREA)
  • Virology (AREA)
  • Immunology (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Genetics & Genomics (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Microbiology (AREA)
  • General Engineering & Computer Science (AREA)
  • Biotechnology (AREA)
  • General Health & Medical Sciences (AREA)
  • Biochemistry (AREA)
  • Analytical Chemistry (AREA)
  • Molecular Biology (AREA)
  • Biophysics (AREA)
  • Physics & Mathematics (AREA)
  • Medicinal Chemistry (AREA)
  • Biomedical Technology (AREA)
  • AIDS & HIV (AREA)
  • Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)

Abstract

L'invention concerne un procédé de routine de dépistage direct et biochimiquement fonctionnel de rétrovirus dans des échantillons biologiques. A cet effet, une extraction spécifique d'une structure, au moyen de ligands immobilisés dirigés contre la surface des virus, est combinée avec une réaction enzymatique, spécifique d'une fonction, de détection de rétrovirus au moyen d'une transcriptase inverse, d'ARNase H, d'intégrase ou de protéase. Pour la réaction enzymatique spécifique du rétrovirus, la transcription inverse au moyen de la transcriptase inverse, d'ARN rétroviral et d'ARNt convient particulièrement. Pendant une étape ultérieure de détection, les produits d'ADNc qui viennent d'être synthétisés sont identifiés par des procédés radioactifs, photométriques, luminescents ou fluorescents. Ce procédé constitue une simulation biochimique du processus naturel d'infection par des rétrovirus et de son évolution dans la cellule (transcription inverse, intégration, maturation) et permet de dépister simultanément plusieurs composants rétroviraux, glycoprotéines de surface, enzymes, protéines structurales, ARN et ARNt. On obtient ainsi un procédé d'une spécificité qualitativement améliorée par rapport aux procédés de l'état de l'art. Le procédé dure 1 à 2 jours, peut être mis en ÷uvre de manière simple et routinière pour réaliser des microtitrages et permet ainsi d'examiner rapidement plusieurs échantillons en même temps. En outre, ce prodédé peut être universellement utilisé pour détecter toutes les espèces de rétrovirus au moyen de ligands spécifiques correspondants. Ce procédé peut être suivi pour dépister des rétrovirus à des fins de diagnostic, de suivi et de thérapie de maladies virales, pour la transfusion et la transplantation médicales, dans la recherche et le développement virologiques et pour le contrôle biologique de la qualité de produits pharmaceutiques et biotechnologiques.
EP94916144A 1993-06-01 1994-05-31 Procede de depistage direct et biochimiquement fonctionnel de retrovirus dans des echantillons biologiques Withdrawn EP0707635A1 (fr)

Applications Claiming Priority (5)

Application Number Priority Date Filing Date Title
DE4318229 1993-06-01
DE4318229 1993-06-01
DE4416300 1994-05-09
DE4416300A DE4416300C2 (de) 1993-06-01 1994-05-09 Verfahren zum direkten und biochemisch funktionellen Nachweis von Retroviren in biologischen Proben
PCT/DE1994/000610 WO1994028115A1 (fr) 1993-06-01 1994-05-31 Procede de depistage direct et biochimiquement fonctionnel de retrovirus dans des echantillons biologiques

Publications (1)

Publication Number Publication Date
EP0707635A1 true EP0707635A1 (fr) 1996-04-24

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ID=25926398

Family Applications (1)

Application Number Title Priority Date Filing Date
EP94916144A Withdrawn EP0707635A1 (fr) 1993-06-01 1994-05-31 Procede de depistage direct et biochimiquement fonctionnel de retrovirus dans des echantillons biologiques

Country Status (5)

Country Link
US (1) US6268123B1 (fr)
EP (1) EP0707635A1 (fr)
JP (1) JPH08510136A (fr)
AU (1) AU6793394A (fr)
WO (1) WO1994028115A1 (fr)

Families Citing this family (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
DE69604051T2 (de) * 1995-01-27 1999-12-16 The Government Of The United States Of America, As Represented By The Secretary Verfahren zum sensitiven nachweis von reverser transcriptase
US7166475B2 (en) * 1999-02-26 2007-01-23 Cyclacel Ltd. Compositions and methods for monitoring the modification state of a pair of polypeptides
US7060506B2 (en) 2000-01-31 2006-06-13 Cyclacel, Ltd. Compositions and methods for monitoring the modification of modification dependent binding partner polypeptides
US20040126900A1 (en) * 2001-04-13 2004-07-01 Barry Stephen E High affinity peptide- containing nanoparticles
WO2002099133A1 (fr) * 2001-05-31 2002-12-12 Fuso Pharmaceutical Industries, Ltd. Procede ameliore de detection et d'identification de micro-organismes causes d'infections
JPWO2002101037A1 (ja) * 2001-05-31 2005-04-07 扶桑薬品工業株式会社 食細胞機能の評価方法およびその利用
US7875422B2 (en) * 2001-06-14 2011-01-25 Cavidi Ab Viral drug susceptibility testing

Family Cites Families (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5077192A (en) * 1988-10-25 1991-12-31 The General Hospital Corporation Method of detecting antigenic, nucleic acid-containing macromolecular entities
DK173091D0 (da) * 1991-10-11 1991-10-11 Schleroseforeningen The Danish Biologisk materiale

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
See references of WO9428115A1 *

Also Published As

Publication number Publication date
US6268123B1 (en) 2001-07-31
WO1994028115A1 (fr) 1994-12-08
AU6793394A (en) 1994-12-20
JPH08510136A (ja) 1996-10-29

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