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


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.


CONCLUSIONS
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.
Bristol Institute for Transfusion Sciences and Histocompatibility and Immunogenetics, NHSBT, Filton, Bristol, UK. marcin.wozniak@nhsbt.nhs.uk
Marcin J
MJ
Woźniak
Claire
C
Bowring
Geoff
G
Lucas
Kay
K
Ridgwell
D005434:Flow Cytometry / Q000379:methods*; D015870:Gene Expression; D057809:HEK293 Cells; D006801:Humans; D007518:Isoantibodies / Q000032:analysis* / Q000097:blood / Q000737:chemistry / Q000276:immunology; D007519:Isoantigens / Q000096:biosynthesis / Q000737:chemistry* / Q000235:genetics / Q000276:immunology; D008562:Membrane Glycoproteins / Q000096:biosynthesis / Q000737:chemistry* / Q000235:genetics / Q000276:immunology; D026901:Membrane Transport Proteins / Q000096:biosynthesis / Q000737:chemistry* / Q000235:genetics / Q000276:immunology; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics / Q000276:immunology; D044967:Serum / Q000737:chemistry* / Q000276:immunology / Q000378:metabolism; D014162:Transfection
C518134:HNA-3a antigen, human; D007518:Isoantibodies; D007519:Isoantigens; D008562:Membrane Glycoproteins; D026901:Membrane Transport Proteins; D011994:Recombinant Proteins; C484524:SLC44A2 protein, human