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Published on: May 19, 2020
Deciphering complement interference in anti-human leukocyte antigen antibody detection with flow beads assays
Jonathan Visentin1, Margaux Vigata, Sophie Daburon
11 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.
Complement activation, specifically C3 cleavage and alternative pathway amplification, interferes with anti-human leukocyte antigen (HLA) antibody detection in flow beads assays by C4 and C3 deposition.
Area of Science:
- Immunology
- Biochemistry
Background:
- Anti-human leukocyte antigen (HLA) antibody detection in solid-phase flow beads assays is crucial for transplantation and autoimmune disease diagnosis.
- Complement activation is a known interference factor, but its precise mechanism in these assays remains unclear.
Purpose of the Study:
- To elucidate the mechanism by which complement activation interferes with anti-HLA antibody detection in Luminex flow beads assays.
- To identify the specific complement components responsible for this interference.
Main Methods:
- Utilized Luminex flow beads screening assay with a pan class I anti-HLA antibody (W6/32).
- Analyzed antibody binding in human sera depleted of specific complement factors (C1q, C4/C3, C2, C3, factor B, C5).
- Detected complement activation and deposition using specific anti-human antibodies (C1q, C4d, C3d).
- Validated findings using clinical samples (patient sera) on HLA single-antigen assays.
Main Results:
- Interference was observed with C2, C3, and factor B-depleted sera, with factor B depletion showing the most pronounced quenching.
- Anti-HLA antibody detection was completely abolished in C5-depleted serum.
- Deposition of C3 and C4 activation products impaired immunoglobulin G and C1q detection.
- C4d detection was hindered by C3 deposition, consistent across different assay formats and patient samples.
Conclusions:
- Complement activation, particularly the deposition of C4 and C3 activation products, is the primary cause of interference in flow beads assays for anti-HLA antibody detection.
- Complete quenching of antibody detection necessitates complement activation via C3 cleavage and amplification through the alternative pathway.
