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Immunoassays

Simultaneous Immunodetection of Anthrax, Plague, and Tularemia from Blood Cultures by Use of Multiplexed Suspension Arrays

Adva Mechaly, Einat Vitner, Haim Levy, Shay Weiss, Elad Bar-David, David Gur, Michal Koren, Hila Cohen, Ofer Cohen, Emanuelle Mamroud, Morly Fisher
Geoffrey A. Land, Editor
Adva Mechaly
Department of Infectious Diseases, Israel Institute for Biological Research, Ness-Ziona, Israel
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Einat Vitner
Department of Infectious Diseases, Israel Institute for Biological Research, Ness-Ziona, Israel
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Haim Levy
Department of Infectious Diseases, Israel Institute for Biological Research, Ness-Ziona, Israel
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Shay Weiss
Department of Infectious Diseases, Israel Institute for Biological Research, Ness-Ziona, Israel
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Elad Bar-David
Department of Infectious Diseases, Israel Institute for Biological Research, Ness-Ziona, Israel
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David Gur
Department of Biochemistry and Molecular Genetics, Israel Institute for Biological Research, Ness-Ziona, Israel
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Michal Koren
Department of Infectious Diseases, Israel Institute for Biological Research, Ness-Ziona, Israel
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Hila Cohen
Department of Biochemistry and Molecular Genetics, Israel Institute for Biological Research, Ness-Ziona, Israel
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Ofer Cohen
Department of Biochemistry and Molecular Genetics, Israel Institute for Biological Research, Ness-Ziona, Israel
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Emanuelle Mamroud
Department of Biochemistry and Molecular Genetics, Israel Institute for Biological Research, Ness-Ziona, Israel
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Morly Fisher
Department of Infectious Diseases, Israel Institute for Biological Research, Ness-Ziona, Israel
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Geoffrey A. Land
Carter BloodCare & Baylor University Medical Center
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DOI: 10.1128/JCM.01479-17
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    FIG 1

    Schematic representation of the microsphere-based assay utilized in this study. The assay consisted of four consecutive steps: coincubation (30 min) of the antigen-containing sample with a suspension of specific antibody-coated Luminex MagPlex beads and biotinylated specific antibodies, leading to the formation of a sandwich immunoassay linked to the magnetic beads (a), separation of unbound components using magnetic force (b), incubation (20 min) of the sandwich immunoassay with SA-R-PE (c), and separation of unbound components using magnetic force (d). The assay was then resuspended in PBS and analyzed in the Luminex MAGPIX instrument.

  • FIG 2
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    FIG 2

    Dose-response curves of multiplexed immunoassays for the detection of spiked soluble disease biomarkers from blood cultures, implementing the 4-plex diagnostic array. Blood cultures (Bactec plus aerobic/F culture vials) containing naive human blood were centrifuged and spiked with recombinant F1 (light blue) and Vag (blue) for plague detection (a) or purified PA (Bordeaux) and soluble anthrax capsular antigen (CAP) (pink) for anthrax detection (b). Points are the averages of at least three independent sets of measurements. Signal-to-noise (S/N) ratios were calculated as described in Materials and Methods, and the limit of detection for each test was determined for S/N ratios to be ≥2 (black dashed line).

  • FIG 3
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    FIG 3

    Detection of native disease biomarkers from inoculated blood cultures utilizing the 4-plex soluble diagnostic array. Blood cultures (Bactec plus aerobic/F culture vials) containing naive human blood were inoculated with Y. pestis EV76 bacteria (a) or B. anthracis Vollum spores (b) and incubated at 37°C. Blood cultures were then centrifuged and plague biomarkers, F1 and Vag, or anthrax biomarkers, PA and capsular antigen, were measured with the soluble 4-plex array. S/N ratios were calculated as described in Materials and Methods, and the limit of detection for each test was determined for S/N ratios to be ≥2 (black dashed line).

  • FIG 4
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    FIG 4

    Blood culture processing procedures for bacterial detection. Blood cultures were processed either with a stepwise centrifugation approach (a) or with a Vacutainer (b). The stepwise centrifugation methodology consisted of a mild centrifugation (140 × g for 10 min) of 1.5 ml of culture fluids. The recovered supernatant (indicated as 1 and highlighted in yellow) was then sedimented (14,000 × g for 5 min), and the pellet (indicated as 2 and highlighted in yellow) was washed with 1 ml of PBS and resuspended in 200 μl of PBS. In the second approach, a 5-ml blood culture sample was centrifuged (1,400 × g for 10 min) in a Vacutainer. The resulting bacterial layer, located on the separating gel, was resuspended in 500 μl of PBS and placed in an Eppendorf tube (indicated as 3 and highlighted in yellow). All bacterium-containing blood fractions that were implemented in the test are indicated in yellow.

  • FIG 5
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    FIG 5

    Evaluation of blood culture fractions for bacterial detection. Blood cultures (Bactec plus aerobic/F culture vials) containing naive human blood were inoculated with Y. pestis EV76 (green) (a) or F. tularensis LVS (red) (b) and incubated at 37°C to a final concentration of 1 × 108 or 1 × 1010 CFU/ml, respectively (determined by viable counts). The resulting blood cultures were processed with the stepwise centrifugation approach or with a Vacutainer. Fractions 1, 2, and 3 (indicated in yellow in Fig. 4) were tested with the 2-plex diagnostic array, undiluted or at 10- and 100-fold dilutions. S/N ratios were calculated as described in Materials and Methods, and the limit of detection for each test was determined for S/N ratios to be ≥2 (black dashed line).

  • FIG 6
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    FIG 6

    Dose-response curves of multiplexed immunoassays for the detection of spiked bacteria from blood cultures implementing the 2-plex diagnostic array. Blood cultures (Bactec plus aerobic/F culture vials) containing naive human blood were mildly centrifuged (140 × g for 10 min) and spiked with inactivated Y. pestis EV76 (green) (a) or inactivated F. tularensis LVS (red) (b). Points are the averages of at least three independent sets of measurements. S/N ratios were calculated as described in Materials and Methods, and the limit of detection for each test was determined for S/N ratios to be ≥2 (black dashed line).

  • FIG 7
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    FIG 7

    Detection of bacteria from inoculated blood cultures utilizing the 2-plex diagnostic array. Blood cultures (Bactec plus aerobic/F culture vials) containing naive human blood were inoculated with Y. pestis EV76 (a) or F. tularensis LVS (b) and incubated at 37°C for different periods. Blood cultures were then processed using the Vacutainer approach, and the presence of Y. pestis (green) or F. tularensis (red) was measured with the 2-plex array. S/N ratios were calculated as described in Materials and Methods, and the limit of detection for each test was determined for S/N ratios to be ≥2 (black dashed line).

Additional Files

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  • Supplemental material

    • Supplemental file 1 -

      Fig. S1 (Dose-response curves of individual Luminex bead-based immunoassays for detection of spiked, soluble disease biomarkers and bacteria) and S2 (Assay performance of fresh versus lyophilized 4-plex diagnostic arrays)

      PDF, 170K

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Simultaneous Immunodetection of Anthrax, Plague, and Tularemia from Blood Cultures by Use of Multiplexed Suspension Arrays
Adva Mechaly, Einat Vitner, Haim Levy, Shay Weiss, Elad Bar-David, David Gur, Michal Koren, Hila Cohen, Ofer Cohen, Emanuelle Mamroud, Morly Fisher
Journal of Clinical Microbiology Mar 2018, 56 (4) e01479-17; DOI: 10.1128/JCM.01479-17

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Simultaneous Immunodetection of Anthrax, Plague, and Tularemia from Blood Cultures by Use of Multiplexed Suspension Arrays
Adva Mechaly, Einat Vitner, Haim Levy, Shay Weiss, Elad Bar-David, David Gur, Michal Koren, Hila Cohen, Ofer Cohen, Emanuelle Mamroud, Morly Fisher
Journal of Clinical Microbiology Mar 2018, 56 (4) e01479-17; DOI: 10.1128/JCM.01479-17
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KEYWORDS

anthrax
immunodetection
magnetic beads
multiplex
tularemia
blood culture
plague

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