Skip Navigation LinksHome > September 27, 2014 - Volume 98 - Issue 6 > Deciphering Complement Interference in Anti–Human Leukocyte...
doi: 10.1097/TP.0000000000000315
Basic and Experimental Research

Deciphering Complement Interference in Anti–Human Leukocyte Antigen Antibody Detection With Flow Beads Assays

Visentin, Jonathan1,2; Vigata, Margaux1; Daburon, Sophie2; Contin-Bordes, Cécile1,2; Fremeaux-Bacchi, Véronique3; Dromer, Claire4; Billes, Marc-Alain5; Neau-Cransac, Martine6; Guidicelli, Gwendaline1; Taupin, Jean-Luc1,2,7

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Background: Anti–human leukocyte antigen (HLA) antibody detection in solid-phase flow beads assays can be quenched by complement activation, but the precise mechanism of this interference is not fully elucidated yet.

Methods: Using the Luminex flow beads screening assay for detection of anti-HLA antibodies, we analyzed the binding of high concentrations of the pan class I anti-HLA monoclonal antibody W6/32 in neat normal, ethylenediaminetetraacetic acid–treated normal and complement factors C1q, C4/C3, C2, C3, factor B or C5-depleted human sera, using anti-mouse immunoglobulin G as the detection antibody. Complement activation and binding to beads were revealed using anti-human C1q, C4d, and C3d antibodies. To translate our findings to the human setting, we used the class I and class II HLA single-antigen flow beads assays and sera from four patients with high titers of antibodies.

Results: Detection of W6/32 did not suffer any interference with C1q and C4/C3–depleted sera. A partial quenching was observed with C2, C3, and factor B-depleted sera, but was more pronounced with the factor B-depleted serum. W6/32 was undetectable in presence of C5-depleted serum. The binding of activation products derived from C3 principally, and also from C4, impaired immunoglobulin G and C1q detection. Accordingly, C4d detection was hindered by deposition of activated C3. Similar findings were obtained with patients’ sera.

Conclusion: Binding of C4 and C3 activation products is the main responsible for complement interference in flow beads assays. A complete quenching requires complement activation through C3 cleavage and its amplification by the alternative pathway.

© 2014 by Lippincott Williams & Wilkins





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