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.
1 Laboratoire d’Immunologie et Immunogénétique, Hôpital Pellegrin, CHU de Bordeaux, Bordeaux, France.
2 UMR CNRS 5164, Université de Bordeaux, Talence, France.
3 Assistance Publique-Hôpitaux de Paris, Hôpital Européen Georges Pompidou, Laboratoire d’immunologie biologique, Paris, France.
4 Service des Maladies Respiratoires, Hôpital Haut-Lévêque, CHU de Bordeaux, Pessac, France.
5 Service de Chirurgie Cardiaque, Hôpital Haut-Lévêque, CHU de Bordeaux, Pessac, France.
6 Service de Transplantation Hépatique, Hôpital Pellegrin, CHU de Bordeaux, Bordeaux, France.
7 Address correspondence to: Jean-Luc Taupin, Pharm. D., Ph.D., Laboratoire d’Immunologie et Immunogénétique, Hôpital Pellegrin, CHU de Bordeaux, Place Amélie Raba Léon, 33076 Bordeaux Cedex, France.
The authors declare no funding or conflicts of interest.
J.T., J.V., and G.G. contributed to the design of the study. J.T. and J.V. participated in the writing of the article. J.V., M.V., S.D., G.G., and J.T. participated in the performance of the research. J.V. and J.T. participated in data analysis. C.C., V.F., C.D., M.B., and M.N. were involved in critical revision of the article.
Received 14 April 2014. Revision requested 9 May 2014.
Accepted 21 May 2014.