Points Antibodies causing FNAIT have decreased Fc fucosylation unlike in refractory

Points Antibodies causing FNAIT have decreased Fc fucosylation unlike in refractory thrombocytopenia. of anti-HPA-1a-specific IgG1 from FNAIT individuals (n = 48) but not in total serum IgG1. Antibodies with a low amount of fucose displayed higher binding affinity to FcγRIIIa and FcγRIIIb but not to FcγRIIa compared with antibodies with a high amount of Fc fucose. As a result these antibodies with a low amount of Fc fucose showed enhanced phagocytosis of platelets using FcγRIIIb+ polymorphonuclear cells or FcγRIIIa+ monocytes as effector cells but not with FcγRIIIa- monocytes. In addition the degree of anti-HPA-1a fucosylation correlated positively with the neonatal platelet counts in FNAIT and negatively to the medical disease severity. In contrast to the FNAIT individuals no changes in core fucosylation were observed for anti-HLA antibodies in refractory thrombocytopenia (post platelet transfusion) indicating that the level of fucosylation may be antigen dependent and/or related to the immune milieu defined by pregnancy. Rabbit Polyclonal to SIN3B. Intro Fetal or neonatal alloimmune thrombocytopenia (FNAIT) is definitely a potentially LAQ824 life-threatening disease where the fetal platelets are targeted by maternal anti-platelet immunoglobulin G (IgG) alloantibodies crossing the LAQ824 placenta. This prospects to IgG-Fc receptor (FcγR)-mediated uptake by phagocytes in the fetal spleen and liver finally resulting in thrombocytopenia.1 Clinical outcome may differ from asymptomatic to petechiae or intracerebral hemorrhage. The strength of the connection between IgG and FcγR depends on several factors including the IgG subclass created during the immune response its relative affinity to FcγRs the manifestation levels of FcγR allotypes FcγR copy number variance cytokines (influencing the manifestation of FcγR) and also the IgG-Fc glycosylation pattern.2 IgG antibodies are glycoproteins containing a branched sugars moiety attached to the Asn297 residue in the Fc part. This glycan is essential for the maintenance of a functional structure and for binding of IgG with FcγR.3-5 In addition the Asn297-linked glycans are substituted with variable amounts of galactose and sialic acid and may additionally carry a bisecting Internet site). Samples were obtained with educated consent from your individuals in accordance with the Declaration of Helsinki. Purification of anti-platelet antibodies from sera HPA-1a-specific alloantibodies were purified in a similar way as explained previously 32 but now instead of eluting from platelets we improved the specificity by eluting the antibodies with formic acid from antigen-coated plates (PAK12; Immucor GTI Diagnostics Waukesha WI). The same plates were utilized for purification of anti-HLA class I antibodies. Further details are explained in the supplemental Methods. Mass spectrometric IgG-Fc glycosylation analysis Nano liquid chromatography-tandem mass spectrometry was performed as explained in the supplemental Methods. Production of recombinant anti-TNP IgG1 antibodies with low and high amounts of LAQ824 Fc fucose The variable regions of the weighty and light chains (VH VL) of the mouse IgG1 anti-2 4 6 (TNP) hapten antibodies were cloned onto human being IgG1 or κ backbone respectively as explained previously 35 and produced in the HEK-293F FreeStyle cell collection expression system (Life Systems Paisley United Kingdom) but now in the presence or absence of 2-deoxy-2-fluoro-l-fucose (2F; Carbosynth Compton Berkshire United Kingdom) to control the level of fucosylation.36 Antibodies were purified on a protein A (WT IgG1) HiTrap HP column (GE Healthcare Life Sciences Little Chalfont United Kingdom) using the Acta Primary Plus system (GE Healthcare Life Sciences) LAQ824 and dialyzed against phosphate-buffered saline (PBS) overnight. IgG-Fc glycosylation was determined by mass spectrometry. Surface plasmon resonance (SPR) SPR measurements were performed with the Biacore 3000 system (Biacore Abdominal Breda The Netherlands) at 25°C. Anti-histidine antibody (GE Healthcare The Netherlands) 25 μg/mL in sodium acetate buffer pH 4.5 (GE Healthcare) was coupled covalently to a CM5 chip (GE Healthcare) following activation with 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide (Sigma-Aldrich Zwijndrecht The Netherlands) 0.4 M in water and = .023 = .043 ρ = 0.320). We also found a significant correlation between the degree of anti-HPA-1a fucosylation and.