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Diagnostic Laboratory Immunology

Measurement of Anti-Erythropoiesis-Stimulating Agent IgG4 Antibody as an Indicator of Antibody-Mediated Pure Red Cell Aplasia

Dohan K. Weeraratne, Andrew J. Kuck, Narendra Chirmule, Daniel T. Mytych
Dohan K. Weeraratne
Department of Clinical Immunology-Medical Sciences, Amgen, Thousand Oaks, California, USA
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Andrew J. Kuck
Department of Clinical Immunology-Medical Sciences, Amgen, Thousand Oaks, California, USA
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Narendra Chirmule
Department of Clinical Immunology-Medical Sciences, Amgen, Thousand Oaks, California, USA
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Daniel T. Mytych
Department of Clinical Immunology-Medical Sciences, Amgen, Thousand Oaks, California, USA
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DOI: 10.1128/CVI.00435-12
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    Fig 1

    Graph representing the signal (in response units) relative to the PNHS spiked with 0.0025 to 100 μg/ml of human anti-ESA antibody 8C10G4. A total of 13 curves were analyzed in duplicate. The graph was constructed using a 4-parameter logistic nonlinear regression model in GraphPad Prism v. 5.0. The response unit per concentration is reported with the standard error of the mean (SEM).

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    Fig 2

    Graphical representation of the measurement of 0, 15, 50, and 200 ng/ml of human antibody 8C10G4 in the presence of 5 μg/ml of anti-ESA antibodies IgG1, IgG2, and IgM as well as a cocktail containing 5 μg/ml each of IgG1, IgG2, and IgM.

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    Fig 3

    Pooled normal human serum samples containing 250 ng/ml of human anti-ESA antibody 8C10G4 spiked with 0 to 100 μg/ml of epoetin alfa. The response unit readout is displayed on the y axis, and the concentration of epoetin alfa (in μg/ml) is displayed on a logarithmic scale on the x axis. The solid line indicates the ACP (243.75).

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    Fig 4

    Graphical representation of a Wilcoxon two-sample test, showing the distribution of the anti-ESA IgG4 concentration (in ng/ml) displayed on the y axis and the patient classification of amPRCA and non-PRCA patients on the x axis.

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  • Table 1

    Results from the SPRIA and ImmunoCAP 100 anti-ESA IgG4 assays and a cell-based assay for neutralizing antibodiesa

    SampleSPRIAImmunoCAP 100Patient classification based on NAb assay
    ResultIsotype(s)Anti-ESA Ab (ng/ml)ResultAnti-ESA IgG4 Ab (ng/ml)
    1PositiveIgG1, IgG2, IgG3, IgG4>10,000Positive145amPRCA
    2NegativeNegativeNon-PRCA
    3PositiveIgG1, IgG2, IgG46,030Positive89amPRCA
    4PositiveIgG1, IgG2, IgG4620Positive89amPRCA
    5PositiveIgG1, IgG2, IgG3, IgG4>10,000Positive669amPRCA
    6NegativeNegativeNon-PRCA
    7PositiveIgG1600NegativeNon-PRCA
    8PositiveIgG1 (IgG4b)1,730Positive95amPRCA
    9NegativeNegativeNon-PRCA
    10NegativeNegativeNon-PRCA
    11NegativeNegativeNon-PRCA
    12NegativeNegativeNon-PRCA
    13PositiveUnable to determine340NegativeNon-PRCA
    14NegativeNegativeNon-PRCA
    15NegativeNegativeNon-PRCA
    16PositiveUnable to determine520Positive59amPRCA
    17PositiveIgG16,860NegativeamPRCA
    18PositiveIgG1840Positive18Non-PRCA
    19PositiveIgG13,690NegativeNon-PRCA
    20PositiveIgM920NegativeNon-PRCA
    21PositiveIgG11,210NegativeNon-PRCA
    22PositiveIgM1.94NegativeNon-PRCA
    23PositiveIgG3700NegativeNon-PRCA
    24PositiveIgG10.93NegativeNon-PRCA
    25PositiveIgG1, IgG2, IgG3, IgG4>10,000Positive4,978amPRCA
    • ↵a All patient samples that had an anti-ESA concentration of greater than 250 ng/ml by SPRIA were further characterized for isotype (IgG1, IgG2, IgG3, IgG4, and IgM) and tested in a cell-based bioassay to determine if neutralizing antibodies were present. All patient samples were tested in the ImmunoCAP anti-ESA IgG4 assay. All samples that that had less than 15 ng/ml of anti-ESA IgG4 were reported as negative.

    • ↵b This patient tested anti-ESA IgG4 antibody positive at a subsequent time point.

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Measurement of Anti-Erythropoiesis-Stimulating Agent IgG4 Antibody as an Indicator of Antibody-Mediated Pure Red Cell Aplasia
Dohan K. Weeraratne, Andrew J. Kuck, Narendra Chirmule, Daniel T. Mytych
Clinical and Vaccine Immunology Jan 2013, 20 (1) 46-51; DOI: 10.1128/CVI.00435-12

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Measurement of Anti-Erythropoiesis-Stimulating Agent IgG4 Antibody as an Indicator of Antibody-Mediated Pure Red Cell Aplasia
Dohan K. Weeraratne, Andrew J. Kuck, Narendra Chirmule, Daniel T. Mytych
Clinical and Vaccine Immunology Jan 2013, 20 (1) 46-51; DOI: 10.1128/CVI.00435-12
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