Calcium‐dependent HLA‐DQ epitope revealed by EDTA mediated inhibition of antibody reactions in the Luminex single antigen bead assay

Calcium‐dependent HLA‐DQ epitope revealed by EDTA mediated inhibition of antibody reactions in the Luminex single antigen bead assay

Complement mediated interference with the detection of antibodies targeting HLA is a known limitation of the single antigen bead (SAB) Luminex assay. Ethylenediaminetetraacetic acid (EDTA) is currently the serum treatment of choice in most histocompatibility laboratories to block complement activati...

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Bibliographic Details
Published in:HLA Vol. 104; no. 2; pp. e15628 - n/a
Main Authors: de Marco, Renato, Noronha, Isaú H., Bottino, Luiza Zainotti Miguel Fahur, Silva, Alan Andrew Santos, Liwski, Robert, Gerbase‐DeLima, Maria
Format: Journal Article
Language:English
Published: Oxford, UK Blackwell Publishing Ltd 01-08-2024
Subjects:
Calcium
calcium‐dependent epitope
Edetic Acid - chemistry
Edetic Acid - pharmacology
EDTA
Epitopes - immunology
Female
Histocompatibility Testing - methods
HLA-DQ Antigens - immunology
HLA‐DQ
Humans
inhibition
Isoantibodies - blood
Isoantibodies - immunology
Kidney Transplantation
Luminex single antigen assay
Middle Aged
HLA‐DQ
inhibition
EDTA
Luminex single antigen assay
calcium‐dependent epitope
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Summary:Complement mediated interference with the detection of antibodies targeting HLA is a known limitation of the single antigen bead (SAB) Luminex assay. Ethylenediaminetetraacetic acid (EDTA) is currently the serum treatment of choice in most histocompatibility laboratories to block complement activation by chelating calcium. The purpose of this study was to investigate a serum with an antibody reactivity to HLA‐DQ6, 7, 8 and 9 molecules, in the Luminex SAB assay, that was inhibited by treatment with EDTA. Serum was from a 55‐year‐old highly sensitised female renal transplant candidate that contained, among others, antibodies to an epitope containing the 74EL eplet, shared by HLA‐DQ6, DQ7, DQ8 and DQ9 molecules. Serum samples were treated with EDTA, dithiothreitol (DTT), or heat prior to testing by SAB assay. EDTA‐treated serum was also tested after the addition of calcium chloride (CaCl2). HLA‐DQ‐specific antibodies were isolated by adsorption/elution method using three informative donor cells and were tested in the absence or presence of EDTA. The antibody reactivity against HLA‐DQ6, DQ7, DQ8 and DQ9 in the SAB assay was significantly inhibited by treating serum and eluates with EDTA and was restored by addition of CaCl2. The study represents the first description of a calcium‐dependent epitope in HLA molecules. The relevance of this finding is that the treatment of sera with EDTA could lead to false‐negative reactions in the SAB assay, which may compromise virtual crossmatching.
ISSN:2059-2302
2059-2310
DOI:10.1111/tan.15628