WO2009109604A1 - Procédé de détection du parvovirus b19 dans le sang ou dans la moelle osseuse - Google Patents

Procédé de détection du parvovirus b19 dans le sang ou dans la moelle osseuse Download PDF

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Publication number
WO2009109604A1
WO2009109604A1 PCT/EP2009/052563 EP2009052563W WO2009109604A1 WO 2009109604 A1 WO2009109604 A1 WO 2009109604A1 EP 2009052563 W EP2009052563 W EP 2009052563W WO 2009109604 A1 WO2009109604 A1 WO 2009109604A1
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WO
WIPO (PCT)
Prior art keywords
parvovirus
cells
kdr
detection
biological sample
Prior art date
Application number
PCT/EP2009/052563
Other languages
English (en)
Inventor
Caroline Schmidt-Lucke
Carsten Tschoepe
Heinz-Peter Schultheiss
Original Assignee
Caroline Schmidt-Lucke
Carsten Tschoepe
Heinz-Peter Schultheiss
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 Caroline Schmidt-Lucke, Carsten Tschoepe, Heinz-Peter Schultheiss filed Critical Caroline Schmidt-Lucke
Publication of WO2009109604A1 publication Critical patent/WO2009109604A1/fr

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Classifications

    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/569—Immunoassay; Biospecific binding assay; Materials therefor for microorganisms, e.g. protozoa, bacteria, viruses
    • G01N33/56983—Viruses
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/005—Assays involving biological materials from specific organisms or of a specific nature from viruses
    • G01N2333/01—DNA viruses
    • G01N2333/015—Parvoviridae, e.g. feline panleukopenia virus, human Parvovirus
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2469/00—Immunoassays for the detection of microorganisms
    • G01N2469/10—Detection of antigens from microorganism in sample from host

Definitions

  • Inflammatory cardiomyopathy can be associated with coronary and peripheral endothelial dysfunction, which has been shown for patients with adenoviral, enteroviral and erythroviral myocardial persistence. Endothelial vasodilator dysfunction predicts long-term disease progression in chronic heart failure and atherosclerosis.
  • the present invention solves the problem by providing a method for the detection of parvovirus B19 in an isolated biological sample, comprising: a) enrichment of KDR+ endothelial progenitor cells (EPC) and/or CD133+ stem cells from the isolated biological sample; b) detection of presence of a parvovirus B19 component in enriched cells.
  • EPC endothelial progenitor cells
  • the present invention is also directed to the use of a method of the invention in:
  • FIG. 1 is a schematic representation of a method of the invention.
  • FIG. 2 shows parvovirus B19 DNA copies per ⁇ g DNA extracted from
  • RNA has been extracted from the enriched cells and subjected to either RT-PCR or Northern analysis.
  • CD34 + cells are subjected to the following protocol for further separation into CD34 + KDR + and CD34 + KDR " .
  • the Dynabeads® were resuspended in 200 ⁇ l Buffer4 and vortexed for 1 second. The tube was placed in the magnet again for 2 minutes and the supernatant was collected containing the target cells. The isolated target cells were washed twice by resuspension in Buffer4 and centrifugation at 30Og for 6 minutes.
  • Buffer! PBS (-Ca2+,-Mg2+)/ 0.1 %BSA, pH -7.4
  • Buffer2 PBS (-Ca2+,-Mg2+)/ 0.1 %BSA, 2mM EDTA, pH -7,4
  • the sample was mixed well and incubated at room temperature for 15 minutes. EasySep® Magnetic Nanoparticles were mixed in order to ensure that they are in a uniform suspension by vigorously pipetting up and down more than 5 times. The particles were added at 50 ⁇ L/mL cells (e.g. for 2 mL of cells, add 100 ⁇ L of nanoparticles). The sample was mixed well and incubated at room temperature for 10 minutes. The cell suspension was topped up to a total volume P632509WO-Zie 12 March 04, 2009
  • the cells were mixed in the tube by gently pipetting up and down 2 - 3 times.
  • the tube (without cap) was placed into the magnet and set aside for 5 minutes.
  • the magnet was picked up and in one continuous motion the magnet and tube were invered, pouring off the supernatant fraction.
  • the magnetically labeled cells remain inside the tube, held by the magnetic field of the EasySep® Magnet.
  • the magnet and tube were left in inverted position for 2 - 3 seconds, then returned to upright position.
  • the tube was removed from the magnet and 2,5 ml_ of recommended medium were added.
  • the cell suspension was mixed by gently pipetting up and down 2 - 3 times.
  • the tube was placed back in the magnet and set aside for 5 minutes. Separation was repeated until a total of three 5-minute separations have been performed in the magnet.
  • the tube was removed from magnet and cells were resuspended in an appropriate amount of desired medium.
  • the CD133 MicroBead Kit formerly termed CD133 Cell Isolation Kit, is a magnetic labeling system designed for the positive selection of CD133 + cells using the CD133 MicroBead Kit (Miltenyi; #130-090-853).
  • the CD133 MicroBead Kit contains FcR Blocking Reagent to avoid unspecific labeling of cells via Fc receptors, and MACS® MicroBeads conjugated to a monoclonal mouse anti- human CD133 antibody (clone AC133) that recognizes epitope CD133/1. The procedure was performed strictly following manufacturers instructions.
  • nPCR erythrovirus DNA by nested PCR
  • ACTB housekeeping gene beta actin
  • Primer sequences were chosen from the sequence of the beta-actin (ACTB) gene. Except for the TaqMan-PCR probe SPV, all primers were obtained from TIB MOLBIOL (Berlin, Germany).
  • Amplification of 664 bp long-PCR fragment for following sequencing of 553 bp-long PCR product was performed by nested- PCR using two new primer pairs. PCR was performed by 35 three-step cycles (45 sec at 95°C, 45 sec at 57°C, 45 sec at 72°C) after initial denaturation for 7 min at 95°C. Nested PCR was performed by 40 cycles with same thermal profile as 1. PCR round.

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  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Immunology (AREA)
  • Engineering & Computer Science (AREA)
  • Virology (AREA)
  • Chemical & Material Sciences (AREA)
  • Biomedical Technology (AREA)
  • Urology & Nephrology (AREA)
  • Molecular Biology (AREA)
  • Hematology (AREA)
  • Microbiology (AREA)
  • Cell Biology (AREA)
  • Biotechnology (AREA)
  • Tropical Medicine & Parasitology (AREA)
  • Food Science & Technology (AREA)
  • Medicinal Chemistry (AREA)
  • Physics & Mathematics (AREA)
  • Analytical Chemistry (AREA)
  • Biochemistry (AREA)
  • General Health & Medical Sciences (AREA)
  • General Physics & Mathematics (AREA)
  • Pathology (AREA)
  • Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)

Abstract

L’invention concerne un procédé de détection du parvovirus B19 dans un échantillon biologique isolé, consistant à : a) enrichir les cellules souches endothéliales (EPC) KDR+ et/ou les cellules indifférenciées CD133+ de l’échantillon biologique isolé ; b) détecter la présence d’un composant du parvovirus B19 dans les cellules enrichies.
PCT/EP2009/052563 2008-03-04 2009-03-04 Procédé de détection du parvovirus b19 dans le sang ou dans la moelle osseuse WO2009109604A1 (fr)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
DE102008012510 2008-03-04
DE102008012510.5 2008-03-04

Publications (1)

Publication Number Publication Date
WO2009109604A1 true WO2009109604A1 (fr) 2009-09-11

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Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/EP2009/052563 WO2009109604A1 (fr) 2008-03-04 2009-03-04 Procédé de détection du parvovirus b19 dans le sang ou dans la moelle osseuse

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WO (1) WO2009109604A1 (fr)

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2012117382A1 (fr) 2011-03-03 2012-09-07 Novartis Ag Procédé de détection d'un antigène parvovirus

Non-Patent Citations (3)

* Cited by examiner, † Cited by third party
Title
KUEHL U ET AL: "ANTIVIRAL INTERFERON THERAPY IN DCM PATIENTS WITH VIRAL PERSISTENCE", CIRCULATION, LIPPINCOTT WILLIAMS & WILKINS, US, vol. 110, no. 17, SUPPL. S, 1 November 2004 (2004-11-01), pages 724, XP009059744, ISSN: 0009-7322 *
SCHMIDT-LUCKE CAROLINE ET AL: "Interferon-beta treatment improves parvovirus B19-induced vascular damage and normalises numbers of CD34+KDR+-progenitors in patients with parvovirus B19 positive positive inflammatory cardiomyopthy", CIRCULATION, vol. 116, no. 16, Suppl. S, October 2007 (2007-10-01), & 80TH ANNUAL SCIENTIFIC SESSION OF THE AMERICAN-HEART-ASSOCIATION; ORLANDO, FL, USA; NOVEMBER 04 -07, 2007, pages 419, XP002531332, ISSN: 0009-7322 *
SCHMIDT-LUCKE CAROLINE ET AL: "Reduced number of circulating endothelial progenitor cells predicts future cardiovascular events - Proof of concept for the clinical importance of endogenous vascular repair", CIRCULATION, vol. 111, no. 22, June 2005 (2005-06-01), pages 2981 - 2987, XP002531336, ISSN: 0009-7322 *

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2012117382A1 (fr) 2011-03-03 2012-09-07 Novartis Ag Procédé de détection d'un antigène parvovirus

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