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Diagnostic Laboratory Immunology

Comparative Study of Different Sources of Pertussis Toxin (PT) as Coating Antigens in IgG Anti-PT Enzyme-Linked Immunosorbent Assays

Aditi Kapasi, Bruce D. Meade, Brian Plikaytis, Lucia Pawloski, Monte D. Martin, Sandra Yoder, Michael T. Rock, Séverine Coddens, Valérie Haezebroeck, Françoise Fievet-Groyne, Garvin Bixler, Charles Jones, Stephen Hildreth, Kathryn M. Edwards, Nancy E. Messonnier, Maria L. Tondella
Aditi Kapasi
Division of Bacterial Diseases, NCIRD, Centers for Disease Control and Prevention, Atlanta, Georgia, USA
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Bruce D. Meade
Meade Biologics LLC, Hillsborough, North Carolina, USA
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Brian Plikaytis
Division of Bacterial Diseases, NCIRD, Centers for Disease Control and Prevention, Atlanta, Georgia, USA
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Lucia Pawloski
Division of Bacterial Diseases, NCIRD, Centers for Disease Control and Prevention, Atlanta, Georgia, USA
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Monte D. Martin
Division of Bacterial Diseases, NCIRD, Centers for Disease Control and Prevention, Atlanta, Georgia, USA
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Sandra Yoder
Department of Pediatrics, Vanderbilt Medical Center, Nashville, Tennessee, USA
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Michael T. Rock
Department of Pediatrics, Vanderbilt Medical Center, Nashville, Tennessee, USA
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Séverine Coddens
GlaxoSmithKline Biologicals, Rixensart, Belgium
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Valérie Haezebroeck
GlaxoSmithKline Biologicals, Rixensart, Belgium
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Françoise Fievet-Groyne
GlaxoSmithKline Biologicals, Rixensart, Belgium
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Garvin Bixler
Sanofi Pasteur, Swiftwater, Pennsylvania, USA
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Charles Jones
Sanofi Pasteur, Swiftwater, Pennsylvania, USA
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Stephen Hildreth
Sanofi Pasteur, Swiftwater, Pennsylvania, USA
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Kathryn M. Edwards
Department of Pediatrics, Vanderbilt Medical Center, Nashville, Tennessee, USA
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Nancy E. Messonnier
Division of Bacterial Diseases, NCIRD, Centers for Disease Control and Prevention, Atlanta, Georgia, USA
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Maria L. Tondella
Division of Bacterial Diseases, NCIRD, Centers for Disease Control and Prevention, Atlanta, Georgia, USA
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DOI: 10.1128/CVI.05460-11
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  • Fig 1
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    Fig 1

    Scatter plots illustrating the comparison of pairs of laboratories for pertussis toxin antigen 1 (PT1). The values plotted are the geometric mean values obtained for the replicate values of samples by a given laboratory using the indicated PT antigen. The green line indicates the Deming regression line for the illustrated comparison. The red line represents the line of identity indicating perfect agreement between the indicated pair of laboratories.

  • Fig 2
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    Fig 2

    Scatter plots illustrating the comparison of pairs of antigens for laboratory A. The values plotted are the geometric mean values obtained for the replicate values of samples by a given laboratory using the indicated pertussis toxin (PT) antigen. The green line indicates the Deming regression line for the illustrated comparison. The red line represents the line of identity indicating perfect agreement between the indicated pair of laboratories.

  • Fig 3
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    Fig 3

    Box plot comparing antigens (1 to 4) for each laboratory. For each laboratory, a consensus value was determined for each sample using the data from that laboratory for all four pertussis toxin (PT) antigens. The relative difference between the consensus and actual values reported by the laboratory was calculated and plotted. This box plot illustrates the distribution of reported results for that laboratory by calculating the log, base 2, of the ratio of each measured value to the consensus value for that sample. For example, a point at 2 on the y axis indicates the value from one antigen within a lab is twice as large as the consensus value. The distributions are plotted separately for each PT antigen. The extremes of the box represent the 25th and 75th percentiles, the middle line represents the median, and the asterisk shows the mean. Vertical lines extend to the most extreme observation that is less than 1.5× the interquartile range (75th to 25th percentiles), and the diamonds and boxes correspond to moderate and severe outlying assay values, respectively. The distance of the mean (asterisk) from the gray dotted line at 0% difference is a direct measure of the mean bias for a given antigen within each laboratory.

  • Fig 4
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    Fig 4

    Box plot comparing laboratories (A to D) for each antigen. For each pertussis toxin (PT) antigen, a consensus value was determined for each sample using the data for that antigen from all four laboratories. The relative difference between the consensus and actual values reported by the laboratory was calculated and plotted. This box plot illustrates the distribution of reported results for that antigen by calculating the log, base 2, of the ratio of each measured value to the consensus value for that sample. For example, a point at 2 on the y axis indicates that the value from one lab for an antigen is twice as large as the consensus value. The distributions are plotted separately for each laboratory. The extremes of the box represent the 25th and 75th percentiles, the middle line represents the median, and the asterisk shows the mean. Vertical lines extend to the most extreme observation that is less than 1.5× the interquartile range (75th to 25th percentiles), and the diamonds and boxes correspond to moderate and severe outlying assay values, respectively. The distance of the mean (asterisk) from the gray dotted line at 0% difference is a direct measure of the mean bias for a given laboratory for each antigen.

  • Fig 5
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    Fig 5

    Results for standard sera (CBER3). This figure illustrates the results from all four laboratories for the CBER3 reference serum included as a control sample. The means and 95% CIs for the ratios of the measured results to the assigned value (200 ELISA units [EU]/ml) are plotted. A plus sign indicates those PT antigens for which the ratio is significantly different than 1.0. The solid black line indicates an observed/expected ratio of 1.0. The broken red lines mark a ratio of plus and minus 1.25-fold, while the broken blue lines mark a ratio of plus and minus 1.50-fold. N is the number of replicate determinations used to generate the confidence intervals.

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    Fig 6

    Results for standard sera (IS). This figure illustrates the results from all four laboratories for the IS reference serum included as a control sample or as a blind test sample, either undiluted or diluted 1:2 in negative serum. The means and 95% CIs for the ratios of the measured results to the assigned value (335 IU/ml) are plotted. A plus sign indicates the PR antigens for which the ratio is significantly different than 1.0. The solid black line indicates an observed/expected ratio of 1.0. The broken red lines mark a ratio of plus and minus 1.25-fold, while the broken blue lines mark a ratio of plus and minus 1.50-fold. N is the number of replicate determinations used to generate the confidence intervals.

Tables

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  • Table 1

    Properties of pertussis toxin antigens from the four sourcesa

    Property of PT antigenList BiologicalsProtein ExpressGlaxoSmithKline BiologicalsSanofi Pasteur
    Production strain165Tohama ITohama I10536
    Purity (%)Not available>99>9599.5
    BioactivityCHO cell assayCHO cell assayCHO cell assayCHO cell assay
    Purity assessment method15% SDS-PAGE13% SDS-PAGESDS-PAGESDS-PAGE/densitometry PT bands
    Storage buffer components50% Glycerol50% Glycerol50% Glycerol50% Glycerol
    0.05 M Tris (pH 7.5)0.05 M Tris-Cl (pH 7.6)25 mM Phosphate buffer (pH 7.6)37.5 mM Potassium phosphate (pH 8.0)
    0.5 M NaCl0.5 M NaCl250 mM NaCl112.5 mM NaCl
    0.01 M Glycine
    Storage temp (°C)−20−20−202–8
    • ↵a PT, pertussis toxin; CHO, Chinese hamster ovary; SDS-PAGE, sodium dodecyl sulfate-polyacrylamide gel electrophoresis.

  • Table 2

    ELISA procedures for all four laboratoriesa

    Assay parameter or reagentCDCVanderbilt Medical CenterSanofi PasteurGlaxoSmithKline Biologicals
    Microtiter plateImmulon 2HBImmulon 2HBNunc MaxiSorpNunc MaxiSorp
    Coating buffer0.05 M carbonate-bicarbonate (pH 9.6)0.05 M carbonate-bicarbonate (pH 9.6)0.05 M carbonate-bicarbonate (pH 9.6)0.05 M carbonate-bicarbonate (pH 9.6)
    Coating incubation time19 h ± 5 h16–24 h18 h ± 2 hOvernight to 3 days
    Coating incubation temp (°C)5 ± 328 (in a humidified chamber)21 ± 35 ± 3
    Washing solutionPBS–0.05% Tween 20PBS–0.05% Tween 20PBS–0.05% Tween 200.9% NaCl–0.05% Tween 20
    Washing cycle300 μl three times250 μl five times300 μl three times with soak300 μl three times
    DiluentPBS–4% BSA–0.05% Tween 20PBS–0.5% BSA–0.5% Tween 20–0.005% PPG0.01 M PBS–10.0% goat serum–0.05% Tween 20PBS–0.05% Tween 20
    Blocking solutionNoneNone0.01 M PBS–1.0% goat serum–0.05% Tween 201% BSA
    Blocking incubation timeNoneNone45 min ± 5 min3 h
    Blocking incubation temp (°C)NoneNone21 ± 337 ± 2
    Serum dilutionSingle point 1:100Eight 2-fold serial dilutionsEight 2-fold serial dilutionsEight 2-fold serial dilutions
    Serum incubation time2 h ± 5 min2 h ± 5 min1 h ± 5 min30 min
    Serum incubation temp (°C)22 ± 428 (in a humidified chamber)37 ± 2RT with shaking
    AntibodyMouse anti-human IgG FcGoat anti-human IgG FcγGoat anti-human IgG FcγGoat anti-human IgG Fc
    ConjugatePeroxidaseAlkaline phosphatasePeroxidasePeroxidase
    Conjugate incubation time2 h ± 5 min16–24 h1 h ± 5 min30 min
    Conjugate incubation temp (°C)22 ± 428 (in a humidified chamber)37 ± 2RT with shaking
    SubstrateTMBpNPPTMBOPD
    Substrate incubation time10 min1 h35 min ± 5 min20 min
    Substrate incubation temp (°C)22 ± 428 (in a humidified chamber)21 ± 3RT
    Wavelength read (nm)450405/590450/540490/620
    Reference standardIn-house pooled seraIn-house pooled seraIn-house pooled seraIn-house pooled sera
    Reference standard calibrationWHO international referenceCBER lot 3CBER lot 3Japanese reference Yoken-H-3
    ControlsNegative, medium, high positiveLow, high positiveNegative, high positiveMedium positive
    Lower limit of quantitation (LOQ)15 IU/ml10 EU/ml4 EU/ml5 EU/ml
    SoftwareGen5/Bio-Tek Instruments, Inc.SoftMax Pro/Molecular DevicesSoftMax Pro/Molecular DevicesSoftMax Pro/Molecular Devices
    Analysis (curve fit)4PL4PLParallel line analysis4PL
    • ↵a PBS, phosphate-buffered saline; BSA, bovine serum albumin; PPG, polypropylene glycol; RT, room temperature; TMB, 3,3′,5,5′-tetramethylbenzidine; pNPP, para-nitrophenylphosphate; OPD, ortho-phenylenediamine dihydrochloride; WHO, World Health Organization; CBER, Center for Biologics Research and Review; IU, international units; EU, ELISA units; 4PL, four-parameter logistic function.

  • Table 3

    Concordance correlation coefficients (rc) for all four antigens

    PT antigenarc between pairs of laboratories for the PT antigen
    PT10.90–0.98
    PT20.93–0.99
    PT30.92–0.98
    PT40.93–0.99
    • ↵a PT, pertussis toxin.

  • Table 4

    Concordance correlation coefficients (rc) for all four laboratories

    Laboratoryrc between pairs of antigens for laboratory
    A0.99
    B0.99–1.00
    C1.00
    D0.97–1.00
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Comparative Study of Different Sources of Pertussis Toxin (PT) as Coating Antigens in IgG Anti-PT Enzyme-Linked Immunosorbent Assays
Aditi Kapasi, Bruce D. Meade, Brian Plikaytis, Lucia Pawloski, Monte D. Martin, Sandra Yoder, Michael T. Rock, Séverine Coddens, Valérie Haezebroeck, Françoise Fievet-Groyne, Garvin Bixler, Charles Jones, Stephen Hildreth, Kathryn M. Edwards, Nancy E. Messonnier, Maria L. Tondella
Clinical and Vaccine Immunology Dec 2011, 19 (1) 64-72; DOI: 10.1128/CVI.05460-11

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Comparative Study of Different Sources of Pertussis Toxin (PT) as Coating Antigens in IgG Anti-PT Enzyme-Linked Immunosorbent Assays
Aditi Kapasi, Bruce D. Meade, Brian Plikaytis, Lucia Pawloski, Monte D. Martin, Sandra Yoder, Michael T. Rock, Séverine Coddens, Valérie Haezebroeck, Françoise Fievet-Groyne, Garvin Bixler, Charles Jones, Stephen Hildreth, Kathryn M. Edwards, Nancy E. Messonnier, Maria L. Tondella
Clinical and Vaccine Immunology Dec 2011, 19 (1) 64-72; DOI: 10.1128/CVI.05460-11
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    • ABSTRACT
    • INTRODUCTION
    • MATERIALS AND METHODS
    • RESULTS
    • DISCUSSION
    • ACKNOWLEDGMENTS
    • FOOTNOTES
    • REFERENCES
  • Figures & Data
  • Info & Metrics
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