title: Deciphering IgM interference in IgG anti-HLA antibody detection with flow beads assays.
abstract: In flow beads assays, the interference of IgM for IgG anti-HLA antibodies detection is not precisely understood. Using the screening flow beads assay for class I HLA antibodies, we analyzed the binding of two IgG mAbs, the anti-class I HLA W6/32 and an anti-beta-2-microglobulin, in the presence of an anti-beta-2-microglobulin IgM mAb. In neat serum, the IgM mAb impaired the detection of both IgG. In EDTA-treated serum, the interference was stronger for the anti-beta-2-microglobulin IgG than for W6/32, in agreement with the finding in surface plasmon resonance that this IgM competed with the anti-beta-2-microglobulin IgG but not with W6/32. The IgM interference was higher in neat than in EDTA-treated serum for both IgG mAbs. The IgM interference was also analyzed with class II single antigen flow beads and sera from two kidney recipients containing IgG and IgM donor specific antibodies. Anti-HLA IgG detection was partially corrected by EDTA, and restored by IgM inactivation with DTT, confirming the results observed with the mAbs. Therefore, three mechanisms can explain the IgM interference for IgG anti-HLA antibodies in flow beads assays: direct competition for antigen, steric hindrance and complement activation.
authors.affiliation: CHU de Bordeaux, Laboratoire d'Immunologie et Immunogénétique, Hôpital Pellegrin, Bordeaux, France; Université de Bordeaux, UMR CNRS, 5164 Talence, France.
authors.forename: Jonathan
authors.initials: J
authors.lastname: Visentin
authors.affiliation: CHU de Bordeaux, Laboratoire d'Immunologie et Immunogénétique, Hôpital Pellegrin, Bordeaux, France.
authors.forename: Gwendaline
authors.initials: G
authors.lastname: Guidicelli
authors.affiliation: Université de Bordeaux, UMR CNRS, 5164 Talence, France; CHU de Bordeaux, Service de Néphrologie, Transplantation, Dialyse, Hôpital Pellegrin, Bordeaux, France.
authors.forename: Lionel
authors.initials: L
authors.lastname: Couzi
authors.affiliation: Université de Bordeaux, UMR CNRS, 5164 Talence, France; CHU de Bordeaux, Service de Néphrologie, Transplantation, Dialyse, Hôpital Pellegrin, Bordeaux, France.
authors.forename: Pierre
authors.initials: P
authors.lastname: Merville
authors.affiliation: One Lambda, Inc., Canoga Park, CA, USA.
authors.forename: Jar-How
authors.initials: JH
authors.lastname: Lee
authors.affiliation: Université de Bordeaux, Laboratoire ARNA, Bordeaux, France; INSERM, U869, Laboratoire ARNA, Pessac, France.
authors.forename: Carmelo
authors.initials: C
authors.lastname: Di Primo
authors.affiliation: CHU de Bordeaux, Laboratoire d'Immunologie et Immunogénétique, Hôpital Pellegrin, Bordeaux, France; Université de Bordeaux, UMR CNRS, 5164 Talence, France. Electronic address: jean-luc.taupin@aphp.fr.
authors.forename: Jean-Luc
authors.initials: JL
authors.lastname: Taupin
mesh_terms: D000906:Antibodies / Q000378:metabolism; D000937:Antigen-Antibody Reactions; D001798:Blood Proteins / Q000378:metabolism*; D003165:Complement System Proteins / Q000378:metabolism; D003951:Diagnostic Errors / Q000517:prevention & control; D005434:Flow Cytometry; D006680:HLA Antigens / Q000276:immunology; D006650:Histocompatibility Testing / Q000379:methods*; D006801:Humans; D007074:Immunoglobulin G / Q000378:metabolism*; D007075:Immunoglobulin M / Q000378:metabolism*; D018189:Immunomagnetic Separation
chemical_list: D000906:Antibodies; D001798:Blood Proteins; D006680:HLA Antigens; D007074:Immunoglobulin G; D007075:Immunoglobulin M; D003165:Complement System Proteins
keywords: Complement; HLA antibodies; IgM; Interference; Single antigen flow beads