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1
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QIAamp nucleic acid purification kit (Qiagen)
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Extraction of nucleic acids from respiratory specimen positive for HBoV by PCR
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18491974
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2
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HotStarTaq DNA polymerase (Qiagen)
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Amplification of the HBoV VP2 gene
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3
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pFastBac vector (Invitrogen)
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Cloning of the HBoV VP2 gene
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4
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Bac-to-Bac Baculovirus Expression System (Invitrogen)
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Generation of recombinant baculovirus
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5
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DH10 Escherichia coli cells
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Transformation for the production of bacmid-containing DNA
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6
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Sf9 insect cells (Invitrogen)
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Transfection with baculovirus for virus-like particle (VLP) production
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7
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Complete Protease Inhibitor Cocktail (Roche Diagnostics)
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Inhibition of protease activity during cell resuspension and lysis
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8
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TE 8.7 buffer (50 mmol/L Tris–HCl and 0.5 mmol/L EDTA [pH 8.7])
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Resuspending Sf9 cells after infection
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9
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Iodixanol gradients
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Purification of virus-like particles (VLPs)
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10
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Beckman SW41 rotor
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Centrifugation of gradients at 150,000 g and 18°C for 18 h
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11
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Sorvall SS-34 rotor
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Centrifugation for clearing cell lysates
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12
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SDS-PAGE gels
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Analysis of gradient fractions
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13
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Philips 410 transmission electron microscope
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Examination of VLPs
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14
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96-well plates (Costar EIA/RIA; Corning)
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Coating with purified VLPs for ELISA
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15
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PBS plus 0.05% Tween 20
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Washing of ELISA wells
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16
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5% nonfat milk (in PBS plus 1% Tween 20)
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Blocking of ELISA wells
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17
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Horseradish peroxidase–conjugated secondary antibody (anti-IgG [H L]; Jackson ImmunoResearch Laboratories)
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Detection of bound primary antibodies in ELISA
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18
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3,3',5,5'-tetramethyl benzidine (Pierce)
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Chromogenic substrate for peroxidase in ELISA
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19
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5N H3PO4
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Stopping the ELISA chromogenic reaction
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20
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DynaTech MR5000 microplate reader
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Measurement of optical densities in ELISA
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21
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Pooled human serum (Bioreclamation)
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Development of the ELISA
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22
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Complete Freund’s adjuvant
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Initial inoculations for HBoV-specific rabbit antisera production
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23
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Incomplete Freund’s adjuvant
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Subsequent inoculations for HBoV-specific rabbit antisera production
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24
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Purified 284-aa B19-VP fusion protein
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Production of B19-specific rabbit antiserum
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25
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Monoclonal antibody against B19 particles (clone 521–50; from Centers for Disease Control and Prevention)
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Usage in immunofluorescence assays
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26
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2.5% paraformaldehyde
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Fixation of cells for immunofluorescence assays
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27
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0.1% Triton X-100
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Permeabilization of cells for immunofluorescence assays
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28
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FITC–conjugated anti–rabbit IgG antibody (Invitrogen)
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Detection of rabbit primary antibodies in immunofluorescence assays
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29
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Texas red–conjugated anti–mouse IgG antibody (Invitrogen)
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Detection of mouse primary antibodies in immunofluorescence assays
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30
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4'-6-diamidino-2-phenylindole (DAPI)
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Nuclear staining in immunofluorescence assays
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31
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EZ-ChIP kit (Upstate)
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Immunoprecipitation of DNA-containing virions
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32
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QIAquick PCR purification units
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Purification of eluted DNA from immunoprecipitation
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33
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Anti-B19 immune rabbit antiserum
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Control in immunoprecipitation assays
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34
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Control nonimmune serum
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Control in immunoprecipitation assays
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35
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Ethidium bromide
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Detection of PCR products by gel electrophoresis
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