A novel enzyme-linked immunosorbent assay method for the detection of human neutrophil antigen-2a antibodies

Authors

  • Behnaz Bayat,

    1. From the Institute for Clinical Immunology and Transfusion Medicine, Justus Liebig University, Giessen, Germany.
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  • Silke Werth,

    1. From the Institute for Clinical Immunology and Transfusion Medicine, Justus Liebig University, Giessen, Germany.
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  • Ulrich J. H. Sachs,

    1. From the Institute for Clinical Immunology and Transfusion Medicine, Justus Liebig University, Giessen, Germany.
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  • Sentot Santoso

    Corresponding author
    1. From the Institute for Clinical Immunology and Transfusion Medicine, Justus Liebig University, Giessen, Germany.
      Sentot Santoso, PhD, Institute for Clinical Immunology and Transfusion Medicine, Justus Liebig University Giessen, Langhansstrasse 7, D-35385 Giessen, Germany; e-mail: sentot.santoso@immunologie.med.uni-giessen.de.
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Sentot Santoso, PhD, Institute for Clinical Immunology and Transfusion Medicine, Justus Liebig University Giessen, Langhansstrasse 7, D-35385 Giessen, Germany; e-mail: sentot.santoso@immunologie.med.uni-giessen.de.

Abstract

BACKGROUND: Antibodies to human neutrophil antigen (HNA)-2a are responsible for a number of immune-mediated neutropenia disorders. Although several methods exist for the identification of anti-HNA-2a, all these methods have several limitations. In this study, a solid-phase enzyme-linked immunosorbent assay (ELISA) using recombinant HNA-2a antigen (rHNA-2a) allowing rapid detection of HNA-2a antibodies was developed.

STUDY DESIGN AND METHODS: Soluble rHNA-2 was generated by transfection of insect cells with CD177 vector. Purified rHNA-2a was immobilized on microtiter wells coated with anti-CD177 and was applied to analyze 10 sera containing HNA-2a antibodies. For the evaluation of the ELISA method, results were compared with the standard assay, MAIGA (monoclonal antibody antigen capture assay) for detection of neutrophil antibodies.

RESULTS: The specificity of HNA-2a antibodies in all sera was confirmed by immunoblotting. Sera were then tested simultaneously in ELISA and MAIGA assays. Nine of 10 sera showed positive reactions in ELISA, whereas only 9 of 10 sera reacted in the standard MAIGA assay. All HNA-2a antibodies were detectable in MAIGA when diluted sera were applied. No reaction was observed with different sera containing neutrophil-reactive antibodies (6 anti-HNA-1a, 4 anti-HNA-1b, and 20 anti-HLA Class I and II) in ELISA. All HNA-2a antibodies were detectable in MAIGA when diluted sera were applied. Notably, sera containing anti-proteinase 3 (PR3) from patients with Wegener's granulomatosis reacted in MAIGA. In contrast, this antibody showed no reaction in ELISA with purified rHNA-2a.

CONCLUSIONS: These results demonstrated that ELISA with rHNA-2a provides a good method for detecting HNA-2a antibodies in human serum. This assay enables to exclude the presence of autoantibody against PR3 in patient's sera, which cannot be differentiated from anti HNA-2a with current serologic methods.

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