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ANTIBODIES AND MEDIATORS OF IMMUNITY

Optimization of the Weck-Cel Collection Method for Quantitation of Cytokines in Mucosal Secretions

Lisa Cencia Rohan, Robert P. Edwards, Lori A. Kelly, Kelly A. Colenello, Frederick P. Bowman, Peggy A. Crowley-Nowick
Lisa Cencia Rohan
Department of Obstetrics, Gynecology and Reproductive Sciences, Magee-Womens Research Institute, University of Pittsburgh, Pittsburgh, Pennsylvania
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Robert P. Edwards
Department of Obstetrics, Gynecology and Reproductive Sciences, Magee-Womens Research Institute, University of Pittsburgh, Pittsburgh, Pennsylvania
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Lori A. Kelly
Department of Obstetrics, Gynecology and Reproductive Sciences, Magee-Womens Research Institute, University of Pittsburgh, Pittsburgh, Pennsylvania
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Kelly A. Colenello
Department of Obstetrics, Gynecology and Reproductive Sciences, Magee-Womens Research Institute, University of Pittsburgh, Pittsburgh, Pennsylvania
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Frederick P. Bowman
Department of Obstetrics, Gynecology and Reproductive Sciences, Magee-Womens Research Institute, University of Pittsburgh, Pittsburgh, Pennsylvania
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Peggy A. Crowley-Nowick
Department of Obstetrics, Gynecology and Reproductive Sciences, Magee-Womens Research Institute, University of Pittsburgh, Pittsburgh, Pennsylvania
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DOI: 10.1128/CDLI.7.1.45-48.2000
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Tables

  • Table 1.

    Mean volume of secretions and calculated dilution factors from mucosal sitesa

    Secretion typeVolume (μl)Dilution
    Cervical29.1 ± 3.022.0 ± 3.4
    Vaginal17.3 ± 3.037.1 ± 3.7
    Oral105 ± 8.17.0 ± 1.3
    • ↵a Volumes and dilutions were calculated based on 40 duplicate samples collected from four women throughout their menstrual cycle. Values are means ± standard errors.

  • Table 2.

    Cytokine recovery from sponges after a 30-min soak at room temperature or 4°Ca

    Protein% Recovery at:
    Room temp4°C
    IL-260.1 ± 5.255.1 ± 4.3
    IL-400
    IL-593.7 ± 12.3488.1 ± 10.9
    IL-634.8 ± 12.640.5 ± 4.4
    IL-1029.6 ± 12.654.2 ± 7.4
    IL-1283.5 ± 11.478.8 ± 5.4
    GM-CSF62.7 ± 10.660.0 ± 3.9
    IFN-γ00
    IgA99.5 ± 10.398.3 ± 5.6
    • ↵a Data are means ± standard deviations from three experiments using one wash with high-salt extraction buffer followed by centrifugation at 16,000 × g.

  • Table 3.

    Cytokine recovery from sponges using final protocol

    ProteinMolecular mass (kDa)a% Recoveryb
    IL-1β17.592.0 ± 5.0
    IL-21585.3 ± 6.9
    IL-415–195.0 ± 0.7
    IL-51890.3 ± 6.8
    IL-620.850.5 ± 2.1
    IL-8858.7 ± 3.7
    IL-1018.755.1 ± 4.3
    IL-127585.9 ± 3.1
    IFN-γ34–500
    GM-CSF18–3263.3 ± 2.0
    IgG150104.0 ± 6.3
    IgA16098.3 ± 5.6
    • ↵a From reference 2.

    • ↵b Determined by comparing ELISA quantitation of extracted cytokine to stock solution of cytokine not absorbed on sponges. Data are means ± standard deviations for three sponges.

  • Table 4.

    Mean cytokine concentrations in cervical secretions

    CytokineAssay detection limita (pg/ml)Cytokine concnb (pg/ml)No. of samples (n = 14) below detection limitc
    Cycle day 9Cycle day 20
    IL-54.014
    IL-60.1046,898.4 ± 3,156.63,255.4 ± 1,198.60
    IL-810.0138,297.1 ± 52,503.654,415.4 ± 11,208.30
    IL-100.028342.7 ± 284.2975.7 ± 864.81
    IL-120.8322.7 ± 123.2387.1 ± 262.11
    • ↵a Level of detection was determined based on product information.

    • ↵b All final concentrations were calculated by using a dilution factor and are means ± standard errors for seven women.

    • ↵c IL-10 was below the assay detection limit on cycle day 9 in one patient, while IL-12 was below the detection limit on cycle day 20.

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Optimization of the Weck-Cel Collection Method for Quantitation of Cytokines in Mucosal Secretions
Lisa Cencia Rohan, Robert P. Edwards, Lori A. Kelly, Kelly A. Colenello, Frederick P. Bowman, Peggy A. Crowley-Nowick
Clinical and Diagnostic Laboratory Immunology Jan 2000, 7 (1) 45-48; DOI: 10.1128/CDLI.7.1.45-48.2000

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Optimization of the Weck-Cel Collection Method for Quantitation of Cytokines in Mucosal Secretions
Lisa Cencia Rohan, Robert P. Edwards, Lori A. Kelly, Kelly A. Colenello, Frederick P. Bowman, Peggy A. Crowley-Nowick
Clinical and Diagnostic Laboratory Immunology Jan 2000, 7 (1) 45-48; DOI: 10.1128/CDLI.7.1.45-48.2000
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