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P083
Abstracts / Human Immunology 75 (2014) 50–141
P083
DETERMINATION OF THE CLINICAL USEFULNESS OF HLA NEUTRALIZATION PROFILES.
Rico Buchli 1, Arend Mulder 2, Annette Jackson 3, Anat R. Tambur 4, René J. Duquesnoy 5, Rebecca D. McAdams 1, Alyson Morris 3, Rick Eggers 6, Georgina Lopez Padilla 6, Daniel Zehnder 7, David P. Lowe 8, David C. Briggs 9, Robert Higgins 10, Frans H.J. Claas 11, Mike Hogan 6, William H. Hildebrand 12. 1 Pure Protein LLC, Oklahoma City, OK, United States; 2 Leiden University Medical Center, Leiden, Netherlands; 3 Johns Hopkins University, Baltimore, MD, United States; 4 Feinberg School of Medicine, Northwestern University, Chicago, IL, United States; 5 University of Pittsburgh Medical Center, Pittsburgh, PA, United States; 6 gmsBiotech, Tucson, AZ, United States; 7 The University of Warwick, Coventry, United Kingdom; 8 Royal Liverpool and Broadgreen University Hospitals NHS Trust, Liverpool, United Kingdom; 9 NHSBT Birmingham, Birmingham, United Kingdom; 10 University Hospitals of Coventry & Warwickshire NHS Trust, Coventry, United Kingdom; 11 Leiden University Medical Center, Leiden, Netherlands; 12 Oklahoma University Health Sciences Center, Oklahoma City, OK, United States. Aim: Some earlier studies indicated that monoclonal Abs can be neutralized and inhibition measured with a bioassay design structured to reduce 95–100% of the maximal signal at 100 lg/ml sHLA if operated in the mid linear range of the response signal. The aim of this investigation was to determine whether the theoretical concepts describing the basic nature of neutralization of HLA antibodies could be applied to more complex serological specimen resulting in clinically useful information. Methods: Sera specimens were tested by individually adding different combinations of sHLA molecules as neutralizing agents prior to response measurement. After subtraction of the resulting inhibitory profile from the non-treated reference profile, a neutralization profile was generated, visualizing Ab responses directly related to the applied neutralizer allele. Results: As shown, sHLA-A⁄02:01 was able to 100% neutralize A2-related responses, leaving other HLA/ antibody interactions untouched. In this example, when also subtracting B⁄08:01 and B⁄49:01, a near complete inhibition of the reference profile was achieved. Minor assay-to-assay background fluctuations of 10% can generally be observed. Significantly, HLA antigens considered directly responsible for the immunizing
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Abstracts / Human Immunology 75 (2014) 50–141
event generally showed a complete inhibition of the reference profile, whereas partial inhibitions were seen as indicator of HLA antibody species present with different antigen/epitope correlation. Conclusion: Creating a neutralizer profile subtracting the inhibitory profile from the reference profile is most helpful for better analytical assessment of signal patterns, delivering a positive view of the blocking event, ultimately allowing better interpretation of antibody populations. As demonstrated, complex reactivity patterns could be reduced to more simplifying outputs. We believe that reductive profiling techniques will greatly assist in obtaining more accurate and meaningful data in the assessment of risk for immunologic rejection.
P083
DETERMINATION OF THE CLINICAL USEFULNESS OF HLA NEUTRALIZATION PROFILES.
Rico Buchli 1, Arend Mulder 2, Annette Jackson 3, Anat R. Tambur 4, René J. Duquesnoy 5, Rebecca D. McAdams 1, Alyson Morris 3, Rick Eggers 6, Georgina Lopez Padilla 6, Daniel Zehnder 7, David P. Lowe 8, David C. Briggs 9, Robert Higgins 10, Frans H.J. Claas 11, Mike Hogan 6, William H. Hildebrand 12. 1 Pure Protein LLC, Oklahoma City, OK, United States; 2 Leiden University Medical Center, Leiden, Netherlands; 3 Johns Hopkins University, Baltimore, MD, United States; 4 Feinberg School of Medicine, Northwestern University, Chicago, IL, United States; 5 University of Pittsburgh Medical Center, Pittsburgh, PA, United States; 6 gmsBiotech, Tucson, AZ, United States; 7 The University of Warwick, Coventry, United Kingdom; 8 Royal Liverpool and Broadgreen University Hospitals NHS Trust, Liverpool, United Kingdom; 9 NHSBT Birmingham, Birmingham, United Kingdom; 10 University Hospitals of Coventry & Warwickshire NHS Trust, Coventry, United Kingdom; 11 Leiden University Medical Center, Leiden, Netherlands; 12 Oklahoma University Health Sciences Center, Oklahoma City, OK, United States. Aim: Some earlier studies indicated that monoclonal Abs can be neutralized and inhibition measured with a bioassay design structured to reduce 95–100% of the maximal signal at 100 lg/ml sHLA if operated in the mid linear range of the response signal. The aim of this investigation was to determine whether the theoretical concepts describing the basic nature of neutralization of HLA antibodies could be applied to more complex serological specimen resulting in clinically useful information. Methods: Sera specimens were tested by individually adding different combinations of sHLA molecules as neutralizing agents prior to response measurement. After subtraction of the resulting inhibitory profile from the non-treated reference profile, a neutralization profile was generated, visualizing Ab responses directly related to the applied neutralizer allele. Results: As shown, sHLA-A⁄02:01 was able to 100% neutralize A2-related responses, leaving other HLA/ antibody interactions untouched. In this example, when also subtracting B⁄08:01 and B⁄49:01, a near complete inhibition of the reference profile was achieved. Minor assay-to-assay background fluctuations of 10% can generally be observed. Significantly, HLA antigens considered directly responsible for the immunizing
107
108
Abstracts / Human Immunology 75 (2014) 50–141
event generally showed a complete inhibition of the reference profile, whereas partial inhibitions were seen as indicator of HLA antibody species present with different antigen/epitope correlation. Conclusion: Creating a neutralizer profile subtracting the inhibitory profile from the reference profile is most helpful for better analytical assessment of signal patterns, delivering a positive view of the blocking event, ultimately allowing better interpretation of antibody populations. As demonstrated, complex reactivity patterns could be reduced to more simplifying outputs. We believe that reductive profiling techniques will greatly assist in obtaining more accurate and meaningful data in the assessment of risk for immunologic rejection.