Detection of HNA-3a and -3b antibodies using transfected cell lines and recombinant proteins

Authors

  • Marcin J. Woźniak,

    Corresponding author
    1. From the Bristol Institute for Transfusion Sciences and Histocompatibility and Immunogenetics, NHSBT, Filton, Bristol, UK.
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  • Claire Bowring,

    1. From the Bristol Institute for Transfusion Sciences and Histocompatibility and Immunogenetics, NHSBT, Filton, Bristol, UK.
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  • Geoff Lucas,

    1. From the Bristol Institute for Transfusion Sciences and Histocompatibility and Immunogenetics, NHSBT, Filton, Bristol, UK.
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    • These authors contributed equally.

  • Kay Ridgwell

    1. From the Bristol Institute for Transfusion Sciences and Histocompatibility and Immunogenetics, NHSBT, Filton, Bristol, UK.
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    • These authors contributed equally.


Marcin J. Woźniak, Bristol Institute for Transfusion Sciences, North Bristol Park, Filton, Bristol BS34 7QH, UK; e-mail: marcin.wozniak@nhsbt.nhs.uk.

Abstract

BACKGROUND: HNA-3 is a diallellic system located on choline transporter-like protein 2 (CTL2), defined by a polymorphism at Amino Acid 154. HNA-3a antibodies are of clinical importance in transfusion-related acute lung injury but antibody detection requires labor-intensive granulocyte isolation from HNA-typed donors and the use of techniques such as the granulocyte agglutination test or granulocyte immunofluorescence test. Also, there is no commercial test for detection of HNA-3 antibodies.

STUDY DESIGN AND METHODS: HEK293 cells were transfected to generate stable cell lines expressing CTL2 fragments (Amino Acids 55-230) and full-length membrane bound CTL2 with HNA-3a and -3b epitopes. Soluble fragments were used in enzyme-linked immunosorbent assays to detect HNA-3 antibodies. The cell lines expressing full-length proteins were trypsin treated to remove HLA antigens and frozen at −80°C. Thawed cells were then used to detect HNA-3 antibodies by flow cytometry.

RESULTS: Glycosylated and soluble CTL2 fragments were correctly recognized by 15 of 31 anti-HNA-3a sera and by both available anti-HNA-3b sera. Twenty-one anti-HLA sera reacted variably with untreated cell lines expressing full-length CTL2. After trypsin treatment of the cell lines, reactivity with HLA antisera was abrogated and all 31 anti-HNA-3a and two anti-HNA-3b sera bound to the corresponding cell line.

CONCLUSION: Whereas soluble, glycosylated CTL2 fragments cannot be used for the detection of HNA-3 antibodies, the HEK293 cells expressing full-length CTL2 proteins were useful in the detection of HNA-3 antibodies even in the presence of HLA antibodies. Moreover, the cell lines can be stored for at least 6 months before use.

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